U.S. patent application number 11/429627 was filed with the patent office on 2008-11-06 for imaging, diagnosis and treatment of disease.
This patent application is currently assigned to Cancer Research Technology Limited, a United Kingdom corporation. Invention is credited to Roy Bicknell, Lukasz Huminiecki.
Application Number | 20080274045 11/429627 |
Document ID | / |
Family ID | 26937319 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274045 |
Kind Code |
A9 |
Bicknell; Roy ; et
al. |
November 6, 2008 |
Imaging, diagnosis and treatment of disease
Abstract
The present invention relates to endothelial cell-specific genes
and encoded polypeptides and materials and uses thereof in the
imaging, diagnosis and treatment of conditions involving the
vascular endothelium.
Inventors: |
Bicknell; Roy; (Headington,
GB) ; Huminiecki; Lukasz; (Hinxton, GB) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
Cancer Research Technology Limited,
a United Kingdom corporation
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20070025913 A1 |
February 1, 2007 |
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Family ID: |
26937319 |
Appl. No.: |
11/429627 |
Filed: |
May 4, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10416090 |
Oct 15, 2003 |
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PCT/GB01/04906 |
Nov 6, 2001 |
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11429627 |
May 4, 2006 |
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60245566 |
Nov 6, 2000 |
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60273662 |
Mar 7, 2001 |
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Current U.S.
Class: |
424/1.49 ;
424/155.1; 435/320.1; 435/326; 435/6.14; 435/7.23; 530/388.8;
530/391.1; 536/23.53 |
Current CPC
Class: |
C07K 14/70503 20130101;
C07K 16/2803 20130101; A61P 9/00 20180101; A61P 35/00 20180101;
C12Q 2600/106 20130101; C12Q 2600/136 20130101; A61P 9/14 20180101;
A61P 35/04 20180101; A61P 15/00 20180101; A61P 27/12 20180101; C12Q
1/6886 20130101; A61P 9/10 20180101; A61P 3/10 20180101; A61P 17/06
20180101; A61K 9/127 20130101; A61P 43/00 20180101; A61K 2039/505
20130101; A61P 41/00 20180101 |
Class at
Publication: |
424/001.49 ;
424/155.1; 435/006; 435/007.23; 435/320.1; 435/326; 530/388.8;
530/391.1; 536/023.53 |
International
Class: |
A61K 51/00 20060101
A61K051/00; C12Q 1/68 20060101 C12Q001/68; G01N 33/574 20060101
G01N033/574; C07H 21/04 20060101 C07H021/04; A61K 39/395 20060101
A61K039/395; C07K 16/46 20070101 C07K016/46; C07K 16/30 20070101
C07K016/30 |
Claims
1. A compound comprising (i) a moiety which selectively binds the
polypeptide ECSM4 and (ii) a further moiety.
2. A compound comprising (i) a moiety which selectively binds the
polypeptide ECSM1 and (ii) a further moiety.
3. A compound according to either claim 1 or 2 wherein the moiety
which selectively binds is an antibody.
4. A compound according to either claim 1 or 2 wherein the moiety
which selectively binds is a peptide.
5. A compound according to either claim 1 or claim 2 wherein the
further moiety is a readily detectable moiety.
6. A compound according to either claim 1 or claim 2 wherein the
further moiety is a directly or indirectly cytotoxic moiety.
7. A compound according to claim 6 wherein the cytotoxic moiety is
a directly cytotoxic chemotherapeutic agent.
8. A compound according to claim 6 wherein the cytotoxic moiety is
a directly cytotoxic polypeptide.
9. A compound according to claim 6 wherein the cytotoxic moiety is
a moiety which is able to convert a relatively non-toxic prodrug
into a cytotoxic drug.
10. A compound according to claim 6 wherein the cytotoxic moiety is
a radiosensitizer.
11. A compound according to either claim 1 or claim 2 wherein the
further moiety comprises a nucleic acid molecule.
12. A compound according to claim 11 wherein the nucleic acid
molecule is a cytotoxic nucleic acid.
13. A compound according to claim 12 wherein the nucleic acid
molecule encodes a directly or indirectly cytotoxic
polypeptide.
14. A compound according to claim 12 wherein the nucleic acid
molecule is directly cytotoxic.
15. A compound according to claim 12 wherein the nucleic acid
encodes a therapeutic polypeptide.
16. A compound according to claim 7 wherein the cytotoxic moiety
comprises a radioactive atom.
17. A compound according to claim 16 wherein the radioactive atom
is any one of phosphorus-32, iodine-125, iodine-131, indium-111,
rhenium186, rhenium-188 or yttrium-90.
18. A compound according to claim 5 wherein the readily detectable
moiety comprises a radioactive atom.
19. A compound according to claim 18 wherein the radioactive atom
is selected from any one of technitium-99m or iodine-123.
20. A compound according to claim 5 wherein the readily detectably
moiety comprises a suitable amount of any one of iodine-123,
iodine131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
21. A compound according to either claim 1 or claim 2 wherein the
further moiety is able to bind selectively to a directly or
indirectly cytotoxic moiety.
22. A compound according to either claim 1 or claim 2 wherein the
further moiety is able to bind selectively to a readily detectable
moiety.
23. A compound according to either claim 1 or claim 2 wherein the
selective binding moiety and the further moiety are polypeptides
which are fused.
24. A nucleic acid molecule encoding a compound according to claim
23.
25. A method of imaging vascular endothelium in the body of an
individual the method comprising administering to the individual an
effective amount of a compound according to claim 5.
26. A method according to claim 25 wherein the vasculature is
neovasculature.
27. A method of diagnosing or prognosing in an individual a
condition which involves the vascular endothelium the method
comprising administering to the individual an effective amount of a
compound according to claim 5.
28. A method according to claim 27 further comprising the step of
detecting the location of the compound in the individual.
29. A method according to either claim 25 or claim 27 wherein the
individual has cancer.
30. A method of treating an individual in need of treatment, the
method comprising administering to the individual an effective
amount of a compound according to either claim 1 or claim 2.
31. A method according to claim 30 wherein the individual in need
of treatment has a proliferative disease or a condition involving
the vascular endothelium such as any one of cancer, psoriasis,
diabetic retinopathy, artherosclerosis or menorrhagia.
32. A method of introducing genetic material selectively into
vascular endothelial cells the method comprising contacting the
cells with a compound according to claim 11.
33-36. (canceled)
37. A polypeptide comprising or consisting of a fragment or variant
or fusion of the ECSM4 polypeptide or a fusion of said fragment or
variant provided that it is not a polypeptide consisting of the
amino acid sequence given between residues 49 and 466 of FIG.
4.
38. A polypeptide according to claim 37 comprising or consisting of
the sequence LSQSPGAVPQALVAWRA, DSVLTPEEVALCLEL, TYGYISVPTA,
KGGVLLCPPRPCLTPT, WLADTW, WLADTWRSTSGSRD, SPPTTYGYIS,
GSLANGWGSASEDNAASARASLVSSSDGSFLAD or FARALAVAVD or a fragment
thereof of at least 5 amino acids.
39. A polypeptide according to claim 37 comprising or consisting of
a sequence present in the human ECSM4 but absent from the
mouseECSM4 sequence.
40. A polypeptide according to claim 39 wherein the sequence
consists or comprises the sequence GGDSLLGGRGSL,
LLQPPARGHAHDGQALSTDL, EPQDYTEPVE, TAPGGQGAPWAEE or
ERATQEPSEHGP.
41. A polypeptide comprising or consisting of the ECSM1 polypeptide
or a fragment or variant or fusion thereof or a fusion of said
fragment or variant.
42. A polynucleotide encoding a polypeptide according to claim 37,
or the complement thereof or a polynucleotide which selectively
hybridises to either of these which polynucleotide is not any one
of the clone or cDNA corresponding to GenBank Accession No AK000805
or the ESTs whose GenBank Accession Nos are listed in Table 11 or
Table 12.
43. A polynucleotide according to claim 42 which encodes a
polypeptide comprising an amino acid sequence with at least 65%
identity to the amino acid sequence given in FIG. 4 or FIG. 7.
44. A polynucleotide encoding a polypeptide according to claim 41
or the complement thereof or a polynucleotide which selectively
hybridises to either of these provided that the polynucleotide is
not one present in ATCC deposit No 209145 or the clone
corresponding to GenBank Accession No AC011526 or the ESTs whose
GenBank Accession Nos are listed in Table 10.
45. A polynucleotide according to claim 45 which encodes a
polypeptide with at least 90% identity to the amino acid sequence
given in FIG. 2.
46. A polynucleotide according to claim 42 which is detectably
labelled.
47. An expression vector comprising a polynucleotide according to
claim 42.
48. A recombinant host cell comprising a polynucleotide according
to claim 42.
49. A recombinant host cell according to claim 48 which is a
bacterial cell.
50. A recombinant host cell according to claim 48 which is a
mammalian cell.
51. A method of producing a polypeptide according to comprising
expressing a polynucleotide according to claim 42.
52. An antibody capable of selectively binding to the ECSM4
polypeptide or the ECSM1 polypeptide.
53. An antibody according to claim 52 which selectively binds a
polypeptide comprising the amino acid sequence located between
residues 1 and 467 of FIG. 12.
54. An antibody according to claim 53 which selectively binds any
one of the amino acid sequences GGDSLLGGRGSL, LLQPPARGHAHDGQALSTDL,
EPQDYTEPVE, TAPGGQGAPWAEE or ERATQEPSEHGP.
55. An antibody according to claim 52 which selectively binds a
polypeptide comprising the amino acid sequence located between
residues 1 and 467 of FIG. 12.
56. An antibody according to claim 52 which is a monoclonal
antibody.
57. An antibody according to claim 52 which is detectably
labelled.
58. A method of detecting endothelial damage or activation in an
individual comprising obtaining a fluid sample from the individual
and detecting the presence of fragments ECSM1 or ECSM4 in the
sample.
59. A method of detecting a tumour or tumour neovasculature or
cardiac disease or endometriosis or artherosclerosis in an
individual comprising obtaining a fluid sample from the individual
and detecting the presence of fragments of ECSM1 or ECSM4 in the
sample.
60. (canceled)
61. A method according to claim 58 wherein endothelial damage is
diagnostic of cancer, cardiac disease, endometriosis or
artherosclerosis in the individual.
62. A method according to claim 58 wherein the individual is one
receiving treatment for cancer, cardiac disease, endometriosis or
artherosclerosis and the amount of fragments of CSM1 or ECSM4 in
the sample is determined and compared either to that in a sample
from an individual who does not have cancer, cardiac disease,
endometriosis or artherosclerosis and/or to the amount in a sample
from the individual prior to commencement of said treatment and the
comparison indicates the efficacy of treatment of the
individual.
63. A method of modulating angiogenesis in an individual, the
method comprising administering to the individual ECSM4, a peptide
or fragment of ECSM4 or a ligand of ECSM4 or an antibody which
selectively binds to ECSM4 or ECSM1.
64. A method of diagnosing a condition which involves aberrant or
excessive growth of vascular endothelium in an individual
comprising obtaining a sample containing nucleic acid from the
individual and contacting said sample with a polynucleotide which
selectively hybridises to a nucleic acid which encodes the ECSM4
polypeptide or the ECSM4 polypeptide.
65. A method of reducing the expression of the ECSM4 or ECSM1
polynucleotide in an individual, comprising administering to the
individual an agent which selectively prevents expression of ECSM4
or ECSM1.
66. A method according to claim 65 wherein the agent is an
antisense nucleic acid.
67. A method of screening for a molecule that binds to ECSM4 or a
suitable variant, fragment or fusion thereof, or a fusion of a said
fragment or fusion thereof, the method comprising 1) contacting a)
the said polypeptide with b) a test molecule, 2) detecting the
presence of a complex containing the ECSM4 polypeptide and a test
molecule and optionally 3) identifying any test molecule bound to
the ECSM4 polypeptide.
68. A polynucleotide comprising a promoter and/or regulatory
portion of either of the ECSM1 or ECSM4 genes.
69. A polynucleotide according to claim 68 which has
transcriptional promoter activity selective to endothelial
cells.
70. A polynucleotide according to either one of claims 68 or 69
which is regulated by hypoxic conditions.
71. A polynucleotide according to claim 68 operatively linked to a
polynucleotide encoding a polypeptide.
72. A polynucleotide according to claim 71 wherein the polypeptide
is a therapeutic polypeptide.
73. A polynucleotide according to claim 72 wherein the polypeptide
is a therapeutic polypeptide suitable for treating a hypoxic
condition such as cancer, cardiac disease, endometriosis or
artherosclerosis.
74. A polynucleotide according to claim 68 which is suitable for
use in gene therapy.
75. A kit of parts comprising a compound according to claim 8 and a
relatively non-toxic prodrug.
76. A kit of parts comprising a compound according to claim 20 and
any one of a directly or indirectly cytotoxic moiety or a readily
detectable moiety to which the said compound is able to bind via
its further moiety.
77-78. (canceled)
79. A method of treating an individual with cancer, cardiac
disease, a hypoxic condition, endometriosis or artherosclerosis
comprising administering to the individual a polynucleotide
according to claim 68.
80. A method of modulating angiogenesis in an individual comprising
administering to the individual a polynucleotide according claims
68 or a polynucleotide which is capable of expressing ECSM4 or a
fragment or variant thereof or which comprises an ECSM4 antisense
nucleic acid.
81-83. (canceled)
84. A transgenic non-human mammal comprising a transgene which
encodes a polypeptide according to claim 37.
85. A non-human mammal wherein if it contains an ECSM1 gene or an
ECSM4 gene the gene or genes are missing or mutated.
86. A non-human mammal according to either one of claims 84 or 85
which is a rodent, preferably mouse.
Description
[0001] The present invention relates to genes whose expression is
selective for the endothelium and use of these genes or gene
products, or molecules which bind thereto, in imaging, diagnosis
and treatment of conditions involving the vascular endothelium.
[0002] The endothelium plays a central role in many physiological
and pathological processes and it is known to be an exceptionally
active transcriptional site. Approximately 1,000 distinct genes are
expressed in an endothelial cell. In contrast red blood cells were
found to express 8, platelets 22 and smooth muscle 127 separate
genes (Adams et al, 1995). Known endothelial specific genes attract
much attention from both basic research and the clinical community.
For example, the endothelial specific tyrosine kinases Tie,
TIE2/TEK, KDR, and flt1 are crucial players in the regulation of
vascular integrity, endothelium-mediated inflammatory processes and
angiogenesis (Sato et al, 1993, Sato et al, 1995, Fong et al, 1995,
Shalaby et al, 1995, Alello et al, 1995). Angiogenesis is now
widely recognised as a rate-limiting process for the growth of
solid tumours. It is also implicated in the formation of
atherosclerotic plaques and restenosis. Finally endothelium plays a
central role in the complex and dynamic system regulating
coagulation and hemostasis.
[0003] Of the many distinct genes expressed in an endothelial cell,
not all are entirely endothelial cell selective and so the genes
and their products, and molecules which bind thereto are not
generally useful in the imaging, diagnosis and treatment of
disease. Thus, there remains a need for endothelial cell specific
or selective molecules.
[0004] We report here identification of two highly endothelial
selective genes which we have called: endothelial cell-specific
molecule 1 (ECSM1) and magic roundabout (endothelial cell-specific
molecule 4; ECSM4). The terms ECSM1 and ECSM4 are also used to
indicate, as the context will make clear, the cDNA and polypeptides
encoded by the genes. These genes, and especially ECSM4, are
surprisingly specific in their cell expression profile. ECSM4, for
example, shows similar endothelial-cell selectivity to the marker
currently accepted in the art as the best endothelial cell marker
(von Willibrand Factor). Clearly, such a high level of endothelial
cell specificity is both unprecedented and unexpected.
[0005] ECSM1 (UniGene entry Hs.13957) has no protein or nucleotide
homologues. It is most likely to code for a small protein of 103 aa
(the longest and most up-stream open reading frame which was
identified in the contig sequence). ECSM1 contains two sequence
tagged sites which are unique and definite within the genome (STS
sites; dbSTS G26129 and G28043) and localise to chromosome 19. A
polynucleotide comprising the complement of part of the ECSM1 gene
is described in WO 99/06423 (Human Genome Sciences) (termed "gene
22"; page 31-32) as being expressed primarily in umbilical cord
endothelial cells and to a lesser extent in human adipose tissue.
However, WO 99/06423 discloses an open reading frame (ORF) in the
polynucleotide which encodes a polypeptide of only 45 amino acids.
According to our analyses, this does not represent the correct
polypeptide of 103 amino acids, as the actual start codon in ECSM1
is further 5' than the one identified in WO 99/06423.
[0006] The human magic roundabout (ECSM4) cDNA clone with a long
ORF of more than 417 aa (GenBank Accession No AK000805) and
described in WO 99/46281 as a 3716 nucleotide sequence was
identified by BLAST searches for the Hs.111518 contig. This
sequence is rich in prolines and has several regions of low amino
acid complexity. BLAST PRODOM search (protein families database at
HGMP, UK) identified a 120 bp region of homology to the cytoplasmic
domain conserved family of transmembrane receptors involved in
repulsive axon guidance (ROBO1 DUTT1 protein family, E=4e-07).
Homology was extended to 468 aa (E=1.3e-09) when a more rigorous
analysis was performed using ssearch (Smith and Waterman 1981) but
the region of similarity was still contained to the cytoplasmic
domain. The ROBO1 DUTT1 family comprises the human roundabout
homologue 1 (ROBO1), the mouse gene DUTT1 and the rat ROBO1 (Kidd
et al, 1998, Brose et al, 1999). Because of this region of homology
we called the gene represented by Hs. 111518 "magic roundabout"
(ECSM4). Additionally, BLAST SBASE (protein domain database at
HGMP) suggested a region of similarity to the domain of the
intracellular neural cell adhesion molecule long domain form
precursor (E=2e-11). It should be noted that the true protein
product for magic roundabout is likely to be larger than the 417 aa
coded in the AK000805 clone since the ORF has no apparent up-stream
limit, and size comparison to human roundabout 1 (1651 aa) suggests
a much bigger protein. This is confirmed in FIG. 3 which shows the
translation product of human ECSM4 to be around 118 kDa. However,
ECSM4 is smaller than other members of the roundabout family,
sharing only two of the five Ig domains and two of the three
fibronectin domains in the extracellular region. The intracellular
putative proline rich region that is homologous to those in
roundabout are thought to couple to c-ab1. FIG. 12 shows the full
length amino acid sequence of human ECSM4 (1105 aa), and the
sequence of the mouse homologue is shown in FIG. 13. Nucleotide
coding sequences which display around 99% identity to the ECSM4
nucleotide sequence given in FIG. 12 are disclosed in WO 99/11293
and WO 99/53051.
[0007] Additional sequences which display homology to the ECSM4
polypeptide or polynucleotide sequence are disclosed in EP 1 074
617, WO 00/53756, WO 99/46281, WO 01/23523 and WO 99/11293.
However, none of these publications disclose that the sequences are
selectively expressed in the vascular endothelium, nor suggest that
they may be so expressed.
[0008] Recently intriguing associations between neuronal
differentiation genes and endothelial cells have been discovered.
For example, a neuronal receptor for vascular endothelial growth
factor (VEGF) neuropilin 1 (Soker et al, 1998) was identified. VEGF
was traditionally regarded as an exclusively endothelial growth
factor. Processes similar to neuronal axon guidance are now being
implicated in guiding migration of endothelial cells during
angiogenic capillary sprouting. Thus ephrinB ligands and EphB
receptors are involved in demarcation of arterial and venous
domains (Adams et al, 1999). It is possible that magic roundabout
(ECSM4) may be an endothelial specific homologue of the human
roundabout 1 involved in endothelial cell repulsive guidance,
presumably with a different ligand since similarity is contained
within the cytoplasmic i.e. effector region and guidance receptors
are known to have highly modular architecture (Bashaw and Goodman
1999).
[0009] However, to date there has been no mention of the existence
of an endothelial counterpart, nor the expression pattern of the
magic roundabout (ECSM4) gene being restricted to endothelial cells
especially angiogeneic endothelial cells, nor of any function of
the encoded polypeptide.
[0010] It should be noted that a surprising result of our RT-PCR
analysis, described in Example 1, was that genes identified here
appear to show endothelial specificity (FIG. 1) comparable with the
classic endothelial marker von Willebrand factor (vWF). Expression
of known endothelial specific genes is not usually 100% restricted
to the endothelial cell. Data presented herein shows the quite
unanticipated finding that ECSM4 is not expressed at detectable
levels (at least using the methods described in the examples) in
cell types other than endothelial cells, given the less than 100%
selectivity of known endothelial cell markers. Ribonuclease
protection analysis has confirmed and extended this observation
(FIG. 14a). ECSM4 expression was seen to be restricted to
endothelium (three different isolates) and absent from fibroblast,
carcinoma and neuronal cells. KDR and FLT1 are both expressed in
the male and female reproductive tract: on spermatogenic cells
(Obermair et al, 1999), trophoblasts, and in decidua (Clark et al,
1996). KDR has been shown to define haematopoietic stem cells
(Ziegler et al, 1999). FLT1 is also present on monocytes. In
addition to endothelial cells vWF is strongly expressed in
megakaryocytes (Sporn et al, 1985, Nichols et al, 1985), and in
consequence present on platelets. Similarly, multimerin is present
both in endothelial cells (Hayward et al, 1993) and platelets
(Hayward et al, 1998).
[0011] Generally speaking, endothelial and haematopoietic cells
descend from same embryonic precursors: haemangioblasts and many
cellular markers are shared between these two cell lineages (for
review see Suda et al, 2000). Hence, the finding that the genes
ECSM1 and ECSM4 are not expressed in cells other than those of the
vascular endothelium is highly surprising.
[0012] Determination of genes whose expression is selective for the
vascular endothelium allows selective targeting to these cells and
thereby the specific delivery of molecules for imaging, diagnosis,
prognosis, treatment, prevention and evaluation of therapies for
conditions associated with normal or aberrant vascular growth.
[0013] A first aspect of the invention provides a compound
comprising (i) a moiety which selectively binds the polypeptide
ECSM4 and (ii) a further moiety.
[0014] By "the polypeptide ECSM4" we include a polypeptide whose
sequence comprises or consists of the amino acid sequence given in
FIG. 4 or 5 or 7 or 12 or 13 or whose sequence is encoded by the
nucleotide sequence given in FIG. 4 between nucleotides 1 and 1395
or between nucleotides 2 and 948 of FIG. 5 or FIG. 7 or between
nucleotides 71 and 3442 of FIG. 12 or between nucleotides 6 and
3050 of FIG. 13 and natural variants thereof. Preferably, the ECSM4
polypeptide is one whose amino acid sequence comprises the sequence
given in FIG. 4 or FIG. 12.
[0015] By "the polypeptide ECSM4" we include a polypeptide
represented by SEQ ID No 18085 of EP 1 074 617, SEQ ID No 211 of
either WO 00/53756 or WO99/46281, SEQ ID Nos 24-27, 29, 30, 33, 34,
38 or 39 of WO 01/23523, or SEQ ID No 86 of WO 99/11293, or the
polypeptide represented by SEQ ID No 18084 or 5096 of EP 1 074 617,
SEQ ID No 210 of WO 00/53756 or WO 99/46281, or SEQ ID Nos 22, 23,
96 or 98 of WO 01/23523 or SEQ ID No 31 of WO 99/11293.
[0016] By "the polypeptide ECSM4" we also include any naturally
occurring polypeptide which comprises a consecutive 50 amino acid
residue portion or natural variants thereof of the polypeptide
sequence given in FIG. 4 or 5 or 7 or 12 or 13. Preferably, the
polypeptide is a human polypeptide.
[0017] Embodiments and features of this aspect of the invention are
as described in more detail below.
[0018] A second aspect of the invention provides a compound
comprising (i) a moiety which selectively binds the polypeptide
ECSM1 and (ii) a further moiety.
[0019] Preferably, in the first and second aspects of the
invention, the binding moiety and further moiety are covalently
attached.
[0020] By "the polypeptide ECSM1" we include a polypeptide whose
amino acid sequence comprises or consists of the sequence given in
FIG. 2 and natural variants thereof.
[0021] By "the polypeptide ECSM1" we also include any naturally
occurring polypeptides which comprises a consecutive 50 amino acid
residue portion or natural variants thereof of the polypeptide
sequence given in FIG. 2. Preferably, the polypeptide is a human
polypeptide.
[0022] Preferably, the polypeptide ECSM1 amino acid sequence
comprises the sequence given in FIG. 2 but does not comprise the
amino acid sequence encoded by ATCC deposit No 209145 made on Jul.
17, 1997 for the purposes of WO 99/06423.
[0023] By "natural variants" we include, for example, allelic
variants. Typically, these will vary from the given sequence by
only one or two or three, and typically no more than 10 or 20 amino
acid residues. Typically, the variants have conservative
substitutions.
[0024] In a preferred embodiment of the first or second aspects of
the invention, the moiety capable of selectively binding to the
specified polypeptide is an antibody.
[0025] Preferably, an antibody which selectively binds ECSM1 or a
natural variant thereof is not one which binds a polypeptide
encoded by SEQ ID No 32 of WO 99/06423 or encoded by the nucleic
acid of ATCC deposit No 209145 made on Jul. 17, 1997 for the
purposes of WO 99/06423.
[0026] Preferably, an antibody which selectively binds ECSM1 is one
which binds a polypeptide whose amino acid sequence comprises the
sequence given in FIG. 2 or a natural variant thereof but which
polypeptide does not comprise the amino acid sequence encoded by
ATCC deposit No 209145 made on Jul. 17, 1997.
[0027] Preferably, an antibody which selectively binds ECSM4 is one
which selectively binds a polypeptide with the sequence
GGDSLLGGRGSL, LLQPPARGHAHDGQALSTDL, EPQDYTEPVE, TAPGGQGAPWAEE or
ERATQEPSEHGP or a sequence which is located in the extracellular
portion of ECSM4. As described in more detail below, these
sequences represent amino acid sequences which are only found in
the human ECSM4 and are not found in the mouse ECSM4 polypeptide
sequence.
[0028] Preferably, the moiety which selectively binds ECSM4, such
as an antibody, is one which binds a polypeptide whose amino acid
sequence comprises the sequence given in any one of FIGS. 4, 5, 7,
12 or 13 or a natural variant thereof but does not bind the
polypeptide represented by any one of SEQ ID No 18085 of EP 1 074
617, SEQ ID No 211 of either WO 00/53756 or WO99/46281, SEQ ID Nos
24-27, 29, 30, 33, 34, 38 or 39 of WO 01/23523, or SEQ ID No 86 of
WO 99/11293, or encoded by any one of the nucleotide sequences
represented by SEQ ID No 18084 or 5096 of EP 1 074 617, SEQ ID No
210 of WO 00 53756 or WO 99/46281, or SEQ ID Nos 22, 23, 96 or 98
of WO 01/23523 and SEQ ID No 31 of WO 99/11293.
[0029] By "antibody" we include not only whole immunoglobulin
molecules but also fragments thereof such as Fab, F(ab')2, Fv and
other fragments thereof that retain the antigen-binding site.
Similarly the term "antibody" includes genetically engineered
derivatives of antibodies such as single chain Fv molecules (scFv)
and domain antibodies (dAbs). The term also includes antibody-like
molecules which may be produced using phage-display techniques or
other random selection techniques for molecules which bind to ECSM1
or ECSM4.
[0030] The variable heavy (V.sub.H) and variable light (V.sub.L)
domains of the antibody are involved in antigen recognition, a fact
first recognised by early protease digestion experiments. Further
confirmation was found by "humanisation" of rodent antibodies.
Variable domains of rodent origin may be fused to constant domains
of human origin such that the resultant antibody retains the
antigenic specificity of the rodent parented antibody (Morrison et
al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
[0031] That antigenic specificity is conferred by variable domains
and is independent of the constant domains is known from
experiments involving the bacterial expression of antibody
fragments, all containing one or more variable domains. These
molecules include Fab-like molecules (Better et al (1988) Science
240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038);
single-chain Fv (ScFv) molecules where the V.sub.H and V.sub.L
partner domains are linked via a flexible oligopeptide (Bird et al
(1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci.
USA 85, 5879) and single domain antibodies (dAbs) comprising
isolated V domains (Ward et al (1989) Nature 341, 544). A general
review of the techniques involved in the synthesis of antibody
fragments which retain their specific binding sites is to be found
in Winter & Milstein (1991) Nature 349, 293-299.
[0032] By "ScFv molecules" we mean molecules wherein the V.sub.H
and V.sub.L partner domains are linked via a flexible
oligopeptide.
[0033] The advantages of using antibody fragments, rather than
whole antibodies, are several-fold. The smaller size of the
fragments may lead to improved pharmacological properties, such as
better penetration to the target site. Effector functions of whole
antibodies, such as complement binding, are removed. Fab, Fv, ScFv
and dAb antibody fragments can all be expressed in and secreted
from E. coli, thus allowing the facile production of large amounts
of the said fragments.
[0034] Whole antibodies, and F(ab').sub.2 fragments are "bivalent".
By "bivalent" we mean that the said antibodies and F(ab').sub.2
fragments have two antigen combining sites. In contrast, Fab, Fv,
ScFv and dAb fragments are monovalent, having only one antigen
combining site.
[0035] Although the antibody may be a polyclonal antibody, it is
preferred if it is a monoclonal antibody. In some circumstance,
particularly if the antibody is going to be administered repeatedly
to a human patient, it is preferred if the monoclonal antibody is a
human monoclonal antibody or a humanised monoclonal antibody.
[0036] Suitable monoclonal antibodies which are reactive as said
may be prepared by known techniques, for example those disclosed in
"Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press,
1988) and in "Monoclonal Hybridoma Antibodies: Techniques and
Application", SGR Hurrell (CRC Press, 1982). Polyclonal antibodies
may be produced which are polypepcific or monospecific. It is
preferred that they are monospecific.
[0037] Chimaeric antibodies are discussed by Neuberger et al (1998,
8.sup.th International Biotechnology Symposium Part 2,
792-799).
[0038] Suitably prepared non-human antibodies can be "humanised" in
known ways, for example by inserting the CDR regions of mouse
antibodies into the framework of human antibodies.
[0039] The antibodies may be human antibodies in the sense that
they have the amino acid sequence of human anti-ECSM1 or -ECSM4
antibodies but they may be prepared using methods known in the art
that do not require immunisation of humans. For example, transgenic
mice are available which contain, in essence, human immunoglobulin
genes (see Vaughan et-al (1998) Nature Biotechnol. 16, 535-539.
[0040] In an alternative embodiment, the moiety capable of
selectively binding to a polypeptide is a peptide. The ECSM4/magic
roundabout polypeptide shows homology with the Drosophila, mouse
and human roundabout proteins, which are cell surface receptors for
secreted Slit proteins (Li et al (1996) Cell 96:807-818). Any
cognate ligand for ECSM4/magic roundabout which is capable of
selectively binding the region of the polypeptide which is located
extracellularly may be useful. The extracellular region of ECSM4 is
likely to be located within residues 1-467 of the ECSM4 polypeptide
sequence given in FIG. 12. It is believed that certain peptides may
be cognate ligands for ECSM4. Such a peptide will be a suitable
moiety for selectively binding ECSM4/magic roundabout. Peptides
binding ECSM4 can be identified by means of a screen. A suitable
method or screen for identifying peptides or other molecules which
selectively bind ECSM4 may comprise contacting the ECSM4
polypeptide with a test peptide or other molecule under conditions
where binding can occur, and then determining if the test molecule
or peptide has bound ECSM4. Methods of detecting binding between
two moieties are well known in the art of biochemistry, Preferably,
the known technique of phage display is used to identify peptides
or other ligand molecules which bind to ECSM4. An alternative
method includes the yeast two hybrid system.
[0041] Peptides or other agents which selectively bind ECSM4
include those which modulate or block the function of ECSM4.
[0042] Suitable peptides may be synthesised as described in more
detail below.
[0043] The further moiety may be any further moiety which confers
on the compound a useful property with respect to the treatment or
imaging or diagnosis of diseases or other conditions or states
which involve undesirable neovasculature formation. Such diseases
or other conditions or states are described in more detail below.
In particular, the further moiety is one which is useful in killing
or imaging neovasculature associated with the growth of a tumour.
Preferably, the further moiety is one which is able to kill the
endothelial cells to which the compound is targeted.
[0044] In a preferred embodiment of the invention the further
moiety is directly or indirectly cytotoxic. In particular the
further moiety is preferably directly or indirectly toxic to cells
in neovasculature or cells which are in close proximity to and
associated with neovasculature.
[0045] By "directly cytotoxic" we include the meaning that the
moiety is one which on its own is cytotoxic. By "indirectly
cytotoxic" we include the meaning that the moiety is one which,
although is not itself cytotoxic, can induce cytotoxicity, for
example by its action on a further molecule or by further action on
it.
[0046] In one embodiment the cytotoxic moiety is a cytotoxic
chemotherapeutic agent. Cytotoxic chemotherapeutic agents are well
known in the art.
[0047] Cytotoxic chemotherapeutic agents, such as anticancer
agents, include: alkylating agents including nitrogen mustards such
as mechlorethamine (HN.sub.2), cyclophosphamide, ifosfamide,
melphalan (L-sarcolysin) and chlorambucil; ethylenimines and
methylmelamines such as hexamethylmelamine, thiotepa; alkyl
sulphonates such as busulfan; nitrosoureas such as carmustine
(BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin
(streptozotocin); and triazenes such as decarbazine (DTIC;
dimethyltriazenoimidazole-carboxamide); Antimetabolites including
folic acid analogues such as methotrexate (amethopterin);
pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU),
floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine
arabinoside); and purine analogues and related inhibitors such as
mercaptopurine (6-mercaptopurine; 6-MP), thioguanine
(6-thioguanine; TG) and pentostatin (2'-deoxycoformycin). Natural
Products including vinca alkaloids such as vinblastine (VLB) and
vincristine; epipodophyllotoxins such as etoposide and teniposide;
antibiotics such as dactinomycin (actinomycin D), daunorubicin
(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin
(mithramycin) and mitomycin (mitomycin C); enzymes such as
L-asparaginase; and biological response modifiers such as
interferon alphenomes. Miscellaneous agents including platinum
coordination complexes such as cisplatin (cis-DDP) and carboplatin;
anthracenedione such as mitoxantrone and anthracycline; substituted
urea such as hydroxyurea; methyl hydrazine derivative such as
procarbazine (N-methylhydrazine, ME); and adrenocortical
suppressant such as mitotane (o,p'-DDD) and aminoglutethimide;
taxol and analogues/derivatives; and hormone agonists/antagonists
such as flutamide and tamoxifen.
[0048] Various of these agents have previously been attached to
antibodies and other target site-delivery agents, and so compounds
of the invention comprising these agents may readily be made by the
person skilled in the art. For example, carbodiimide conjugation
(Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159;
incorporated herein by reference) may be used to conjugate a
variety of agents, including doxorubicin, to antibodies or
peptides.
[0049] Carbodiimides comprise a group of compounds that have the
general formula R--N.dbd.C.dbd.N--R', where R and R' can be
aliphatic or aromatic, and are used for synthesis of peptide bonds.
The preparative procedure is simple, relatively fast, and is
carried out under mild conditions. Carbodiimide compounds attack
carboxylic groups to change them into reactive sites for free amino
groups.
[0050] The water soluble carbodiimide,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is
particularly useful for conjugating a functional moiety to a
binding moiety and may be used to conjugate doxorubicin to tumor
homing peptides. The conjugation of doxorubicin and a binding
moiety requires the presence of an amino group, which is provided
by doxorubicin, and a carboxyl group, which is provided by the
binding moiety such as an antibody or peptide.
[0051] In addition to using carbodiimides for the direct formation
of peptide bonds, EDC also can be used to prepare active esters
such as N-hydroxysuccinimide (NHS) ester. The NHS ester, which
binds only to amino groups, then can be used to induce the
formation of an amide bond with the single amino group of the
doxorubicin. The use of EDC and NHS in combination is commonly used
for conjugation in order to increase yield of conjugate formation
(Bauminger & Wilchek, supra, 1980).
[0052] Other methods for conjugating a functional moiety to a
binding moiety also can be used. For example, sodium periodate
oxidation followed by reductive alkylation of appropriate reactants
can be used, as can glutaraldehyde cross-linking. However, it is
recognised that, regardless of which method of producing a
conjugate of the invention is selected, a determination must be
made that the binding moiety maintains its targeting ability and
that the functional moiety maintains its relevant function.
[0053] In a further embodiment of the invention, the cytotoxic
moiety is a cytotoxic peptide or polypeptide moiety by which we
include any moiety which leads to cell death. Cytotoxic peptide and
polypeptide moieties are well known in the art and include, for
example, ricin, abrin, Pseudomonas exotoxin, tissue factor and the
like. Methods for linking them to targeting moieties such as
antibodies are also known in the art. The use of ricin as a
cytotoxic agent is described in Burrows & Thorpe (1993) Proc.
Natl. Acad. Sci. USA 90, 8996-9000, incorporated herein by
reference, and the use of tissue factor, which leads to localised
blood clotting and infarction of a tumour, has been described by
Ran et al (1998) Cancer Res. 58, 4646-4653 and Huang et al (1997)
Science 275, 547-550. Tsai et al (1995) Dis. Colon Rectum 38,
1067-1074 describes the abrin A chain conjugated to a monoclonal
antibody and is incorporated herein by reference. Other ribosome
inactivating proteins are described as cytotoxic agents in WO
96/06641. Pseudomonas exotoxin may also be used as the cytotoxic
polypeptide moiety (see, for example, Aiello et al (1995) Proc.
Natl. Acad. Sci. USA 92, 10457-10461; incorporated herein by
reference).
[0054] Certain cytokines, such as TNF.alpha. and IL-2, may also be
useful as cytotoxic agents.
[0055] Certain radioactive atoms may also be cytotoxic if delivered
in sufficient doses. Thus, the cytotoxic moiety may comprise a
radioactive atom which, in use, delivers a sufficient quantity of
radioactivity to the target site so as to be cytotoxic. Suitable
radioactive atoms include phosphorus-32, iodine-125, iodine-131,
indium-111, rhenium-186, rhenium-188 or yttrium-90, or any other
isotope which emits enough energy to destroy neigbbouring cells,
organelles or nucleic acid. Preferably, the isotopes and density of
radioactive atoms in the compound of the invention are such that a
dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000
cGy) is delivered to the target site and, preferably, to the cells
at the target site and their organelles, particularly the
nucleus.
[0056] The radioactive atom may be attached to the binding moiety
in known ways. For example EDTA or another chelating agent may be
attached to the binding moiety and used to attach .sup.111In or
.sup.90Y; Tyrosine residues may be labelled with .sup.125I or
.sup.131I.
[0057] The cytotoxic moiety may be a suitable indirectly cytotoxic
polypeptide. In a particularly preferred embodiment, the indirectly
cytotoxic polypeptide is a polypeptide which has enzymatic activity
and can convert a relatively non-toxic prodrug into a cytotoxic
drug. When the targeting moiety is an antibody this type of system
is often referred to as ADEPT (Antibody-Directed Enzyme Prodrug
Therapy). The system requires that the targeting moiety locates the
enzymatic portion to the desired site in the body of the patient
(ie the site expressing ECSM1 or ECSM4, such as new vascular tissue
associated with a tumour) and after allowing time for the enzyme to
localise at the site, administering a prodrug which is a substrate
for the enzyme, the end product of the catalysis being a cytotoxic
compound. The object of the approach is to maximise the
concentration of drug at the desired site and to minimise the
concentration of drug in normal tissues (see Senter, P. D. et al
(1988) "Anti-tumor effects of antibody-alkaline phosphatase
conjugates in combination with etoposide phosphate" Proc. Natl.
Acad. Sci. USA 85, 4842-4846; Bagshawe (1987) Br. J. Cancer 56,
531-2; and Bagshawe, K. D. et al (1988) "A cytotoxic agent can be
generated selectively at cancer sites" Br. J. Cancer. 58,
700-703.)
[0058] Clearly, any ECSM1 or ECSM4 binding moiety may be used in
place of an anti-ECSM1 or anti-ECSM4 antibody in this type of
directed enzyme prodrug therapy system.
[0059] The enzyme and prodrug of the system using an ECSM1 or ECSM4
targeted enzyme as described herein may be any of those previously
proposed. The cytotoxic substance may be any existing anti-cancer
drug such as an alkylating agent; an agent which intercalates in
DNA; an agent which inhibits any key enzymes such as dihydrofolate
reductase, thymidine synthetase, ribonucleotide reductase,
nucleoside kinases or topoisomerase; or an agent which effects cell
death by interacting with any other cellular constituent. Etoposide
is an example of a topoisomerase inhibitor.
[0060] Reported prodrug systems include: a phenol mustard prodrug
activated by an E. coli .beta.-glucuronidase (Wang et al, 1992 and
Roffler et al, 1991); a doxorubicin prodrug activated by a human
.beta.-glucuronidase (Bosslet et al, 1994); further doxorubicin
prodrugs activated by coffee bean .alpha.-galactosidase (Azoulay et
al., 1995); daunorubicin prodrugs, activated by coffee bean
.alpha.-D-galactosidase (Gesson et al, 1994); a 5-fluorouridine
prodrug activated by an E. coli .beta.-D-galactosidase (Abraham et
al, 1994); and methotrexate prodrugs (eg methotrexate-alanine)
activated by carboxypeptidase A (Kuefner et al, 1990, Vitols et al,
1992 and Vitols et al, 1995). These and others are included in the
following table. TABLE-US-00001 Enzyme Prodrug Carboxypeptidase G2
Derivatives of L-glutamic acid and benzoic acid mustards, aniline
mustards, phenol mustards and phenylenediamine mustards;
fluorinated derivatives of these Alkaline phosphatase Etoposide
phosphate Mitomycin phosphate Beta-glucuronidase p-Hydroxyaniline
mustard-glucuronide Epirubicin-glucuronide Penicillin-V-amidase
Adriamycin-N phenoxyacetyl Penicillin-G-amidase N-(4'-hydroxyphenyl
acetyl) palytoxin Doxorubicin and melphalan Beta-lactamase Nitrogen
mustard-cephalosporin p-phenylenediamine; doxorubicin derivatives;
vinblastine derivative-cephalosporin, cephalosporin mustard; a
taxol derivative Beta-glucosidase Cyanophenylmethyl-beta-D-gluco-
pyranosiduronic acid Nitroreductase
5-(Azaridin-1-yl-)-2,4-dinitrobenzamide Cytosine deaminase
5-Fluorocytosine Carboxypeptidase A Methotrexate-alanine
[0061] (This table is adapted from Bagshawe (1995) Drug Dev. Res.
34, 220-230, from which full references for these various systems
may be obtained; the taxol derivative is described in Rodrigues, M.
L. et al (1995) Chemistry & Biology 2, 223).
[0062] Suitable enzymes for forming part of the enzymatic portion
of the invention include: exopeptidases, such as carboxypeptidases
G, G1 and G2 (for glutamylated mustard prodrugs), carboxypeptidases
A and B (for MTX-based prodrugs) and aminopeptidases (for
2-.alpha.-aminocyl MTC prodrugs); endopeptidases, such as eg
thrombolysin (for thrombin prodrugs); hydrolases, such as
phosphatases (eg alkaline phosphatase) or sulphatases (eg aryl
sulphatases) (for phosphylated or sulphated prodrugs); amidases,
such as penicillin amidases and arylacyl amidase; lactamases, such
as .beta.-lactamases; glycosidases, such as .beta.-glucuronidase
(for .beta.-glucuronomide anthracyclines), .alpha.-galactosidase
(for amygdalin) and .beta.-galactosidase (for .beta.-galactose
anthracycline); deaminases, such as cytosine deaminase (for 5FC);
kinases, such as urokinase and thymidine kinase (for gancyclovir);
reductases, such as nitroreductase (for CB1954 and analogues),
azoreductase (for azobenzene mustards) and DT-diaphorase (for
CB1954); oxidases, such as glucose oxidase (for glucose), xanthine
oxidase (for xanthine) and lactoperoxidase; DL-racemases, catalytic
antibodies and cyclodextrins.
[0063] The prodrug is relatively non-toxic compared to the
cytotoxic drug. Typically, it has less than 10% of the toxicity,
preferably less than 1% of the toxicity as measured in a suitable
in vitro cytotoxicity test.
[0064] It is likely that the moiety which is able to convert a
prodrug to a cytotoxic drug will be active in isolation from the
rest of the compound but it is necessary only for it to be active
when (a) it is in combination with the rest of the compound and (b)
the compound is attached to, adjacent to or internalised in target
cells.
[0065] When each moiety of the compound is a polypeptide, the two
portions may be linked together by any of the conventional ways of
cross-linking polypeptides, such as those generally described in
O'Sullivan et al (1979) Anal. Biochem. 100, 100-108. For example,
the ECSM1 or ECSM4 binding moiety may be enriched with thiol groups
and the further moiety reacted with a bifunctional agent capable of
reacting with those thiol groups, for example the
N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Amide and
thioether bonds, for example achieved with
m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more
stable in vivo than disulphide bonds.
[0066] Alternatively, the compound may be produced as a fusion
compound by recombinant DNA techniques whereby a length of DNA
comprises respective regions encoding the two moieties of the
compound of the invention either adjacent one another or separated
by a region encoding a linker peptide which does not destroy the
desired properties of the compound. Conceivably, the two portions
of the compound may overlap wholly or partly.
[0067] The DNA is then expressed in a suitable host to produce a
polypeptide comprising the compound of the invention.
[0068] The invention also provides a kit of parts (or a therapeutic
system) comprising (1) a compound of the invention wherein the
further moiety which is able to convert a relatively non-toxic
prodrug into a cytotoxic drug and (2) a relatively non-toxic
prodrug. The kit of parts may comprise any of the compounds of the
invention and appropriate prodrugs as herein disclosed.
[0069] The invention also provides a kit of parts (or a therapeutic
system) comprising (1) a compound of the invention wherein the
further moiety is able to bind selectively to a directly or
indirectly cytotoxic moiety or to a readily detectable moiety and
(2) any one of a directly or indirectly cytotoxic or a readily
detectable moiety to which the further moiety of the compound is
able to bind.
[0070] The cytotoxic moiety may be a radiosensitizer.
Radiosensitizers include fluoropyrimidines, thymidine analogues,
hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated
pyrimidines, 3-aminobenzamide, 3-aminobenzodiamide, etanixadole,
pimonidazole and misonidazole (see, for example, McGinn et al
(1996) J. Natl. Cancer Inst. 88, 1193-11203; Shewach & Lawrence
(1996) Invest. New Drugs 14, 257-263; Horsman (1995) Acta Oncol.
34, 571-587; Shenoy & Sinagh (1992) Clin. Invest. 10, 533-551;
Mitchell et al (1989) Int. J. Radiat. Biol. 56, 827-836; Iliakis
& Kurtzman (1989) Int. J. Radiat. Oncol. Biol. Phys. 16,
1235-1241; Brown (1989) Int. J. Radiat. Oncol. Biol. Phys. 16,
987-993; Brown (1985) Cancer 55, 2222-2228).
[0071] Also, delivery of genes into cells can radiosensitise them,
for example delivery of the p53 gene or cyclin D (Lang et al (1998)
J. Neurosurg. 89, 125-132; Coco Martin et al (1999) Cancer Res. 59,
1134-1140).
[0072] The further moiety may be one which becomes cytotoxic, or
releases a cytotoxic moiety, upon irradiation. For example, the
boron-10 isotope, when appropriately irradiated, releases a
particles which are cytotoxic (see for example, U.S. Pat. No.
4,348,376 to Goldenberg; Primus et al (1996) Bioconjug. Chem. 7,
532-535).
[0073] Similarly, the cytotoxic moiety may be one which is useful
in photodynamic therapy such as photofrin (see, for example,
Dougherty et al (1998) J. Natl. Cancer Inst. 90, 889-905).
[0074] The further moiety may comprise a nucleic acid molecule
which is directly or indirectly cytotoxic. For example, the nucleic
acid molecule may be an antisense oligonucleotide which, upon
localisation at the target site is able to enter cells and lead to
their death. The oligonucleotide, therefore, may be one which
prevents expression of an essential gene, or one which leads to a
change in gene expression which causes apoptosis.
[0075] Examples of suitable oligonucleotides include those directed
at bc1-2 (Ziegler et al (1997) J. Natl. Cancer Inst. 89,
1027-1036), and DNA polymerase a and topoisomerase II.alpha. (Lee
et al (1996) Anticancer Res. 16, 1805-1811.
[0076] Peptide nucleic acids may be useful in place of conventional
nucleic acids (see Knudsen & Nielsen (1997) Anticancer Drugs 8,
113-118).
[0077] In a further embodiment, the binding moiety may be comprised
in a delivery vehicle for delivering nucleic acid to the target.
The delivery vehicle may be any suitable delivery vehicle. It may,
for example, be a liposome containing nucleic acid, or it may be a
virus or virus-like particle which is able to deliver nucleic acid.
In these cases, the moiety which selectively binds to ECSM1 or
ECSM4 is typically present on the surface of the delivery vehicle.
For example, the moiety which selectively binds to ECSM1 or ECSM4,
such as a suitable antibody fragment, may be present in the outer
surface of a liposome and the nucleic acid to be delivered may be
present in the interior of the liposome. As another example, a
viral vector, such as a retroviral or adenoviral vector, is
engineered so that the moiety which selectively binds to ECSM1 or
ECSM4 is attached to or located in the surface of the viral
particle thus enabling the viral particle to be targeted to the
desired site. Targeted delivery systems are also known such as the
modified adenovirus system described in WO 94/10323 wherein,
typically, the DNA is carried within the adenovirus, or
adenovirus-like, particle. Michael et al (1995) Gene Therapy 2,
660-668 describes modification of adenovirus to add a
cell-selective moiety into a fibre protein. Targeted retroviruses
are also available for use in the invention; for example, sequences
conferring specific binding affinities may be engineered into
preexisting viral env genes (see Miller & Vile (1995) Faseb J.
9, 190-199 for a review of this and other targeted vectors for gene
therapy).
[0078] Immunoliposomes (antibody-directed liposomes) may be used in
which the moiety which selectively binds to ECSM1 or ECSM4 is an
antibody. For the preparation of immuno-liposomes MPB-PE
(N-[4-p-maleimidophenyl)butyryl]-phosphatidylethanolamine) is
synthesised according to the method of Martin & Papahadjopoulos
(1982) J. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the
liposomal bilayers to allow a covalent coupling of the anti-ECSM1
or -ECSM4 antibody, or fragment thereof, to the liposomal surface.
The liposome is conveniently loaded with the DNA or other genetic
construct for delivery to the target cells, for example, by forming
the said liposomes in a solution of the DNA or other genetic
construct, followed by sequential extrusion through polycarbonate
membrane filters with 0.6 .mu.m and 0.2 .mu.m pore size under
nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA
construct is separated from free DNA construct by
ultracentrifugation at 80 000.times.g for 45 min. Freshly prepared
MPB-PE-liposomes in deoxygenated buffer are mixed with freshly
prepared antibody (or fragment thereof) and the coupling reactions
are carried out in a nitrogen atmosphere at 4.degree. C. under
constant end over end rotation overnight. The immunoliposomes are
separated from unconjugated antibodies by ultracentrifugation at 80
000.times.g for 45 min. Immunoliposomes may be injected
intraperitoneally or directly into the tumour.
[0079] The nucleic acid delivered to the target site may be any
suitable DNA which leads, directly or indirectly, to cytotoxicity.
For example, the nucleic acid may encode a ribozyme which is
cytotoxic to the cell, or it may encode an enzyme which is able to
convert a substantially non-toxic prodrug into a cytotoxic drug
(this latter system is sometime called GDEPT: Gene Directed Enzyme
Prodrug Therapy).
[0080] Ribozymes which may be encoded in the nucleic acid to be
delivered to the target are described in Cech and Herschlag
"Site-specific cleavage of single stranded DNA" U.S. Pat. No.
5,180,818; Altman et al "Cleavage of targeted RNA by RNAse P" U.S.
Pat. No. 5,168,053, Cantin et al "Ribozyme cleavage of HIV-1 RNA"
U.S. Pat. No. 5,149,796; Cech et al "RNA ribozyme restriction
endoribonucleases and methods", U.S. Pat. No. 5,116,742; Been et al
"RNA ribozyme polymerases, dephosphorylases, restriction
endonucleases and methods", U.S. Pat. No. 5,093,246; and Been et al
"RNA ribozyme polymerases, dephosphorylases, restriction
endoribonucleases and methods; cleaves single-stranded RNA at
specific site by transesterification", U.S. Pat. No. 4,987,071, all
incorporated herein by reference, Suitable targets for ribozymes
include transcription factors such as c-fos and c-myc, and bc1-2.
Durai et al (1997) Anticancer Res. 17, 3307-3312 describes a
hammerhead ribozyme against bc1-2.
[0081] EP 0 415 731 describes the GDEPT system. Similar
considerations concerning the choice of enzyme and prodrug apply to
the GDEPT system as to the ADEPT system described above.
[0082] The nucleic acid delivered to the target site may encode a
directly cytotoxic polypeptide.
[0083] Alternatively, the further portion may comprise a
polypeptide or a polynucleotide encoding a polypeptide which is not
either directly or indirectly cytotoxic but is of therapeutic
benefit. Examples of such polypeptides include anti-proliferative
or anti-inflammatory cytokines which could be of benefit in
artherosclerosis, and anti-proliferative, immunomodulatory or
factors influencing blood clotting may be of benefit in treating
cancer.
[0084] The further moiety may usefully be an inhibitor of
angiogenesis such as the peptides angiostatin or endostatin. The
further moiety may also usefully be an enzyme which converts a
precursor polypeptide to angiostatin or endostatin. Human matrix
metallo-proteases such as macrophage elastase, gelatinase and
stromolysin convert plasminogen to angiostatin (Cornelius et al
(1998) J. Immunol. 161, 6845-6852). Plasminogen is a precursor of
angiostatin.
[0085] In a further embodiment of the invention, the further moiety
comprised in the compound of the invention is a readily detectable
moiety.
[0086] By a "readily detectable moiety" we include the meaning that
the moiety is one which, when located at the target site following
administration of the compound of the invention into a patient, may
be detected, typically non-invasively from outside the body and the
site of the target located. Thus, the compounds of this embodiment
of the invention are useful in imaging and diagnosis.
[0087] Typically, the readily detectable moiety is or comprises a
radioactive atom which is useful in imaging. Suitable radioactive
atoms include technetium-99m or iodine-123 for scintigraphic
studies. Other readily detectable moieties include, for example,
spin labels for magnetic resonance imaging (MRI) such as iodine-123
again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron. Clearly, the compound of
the invention must have sufficient of the appropriate atomic
isotopes in order for the molecule to be readily detectable.
[0088] The radio- or other labels may be incorporated in the
compound of the invention in known ways. For example, if the
binding moiety is a polypeptide it may be biosynthesised or may be
synthesised by chemical amino acid synthesis using suitable amino
acid precursors involving, for example, fluorine-19 in place of
hydrogen. Labels such as .sup.99mTc, .sup.123I, .sup.186Rh,
.sup.188Rh and .sup.111In can, for example, be attached via
cysteine residues in the binding moiety. Yttrium-90 can be attached
via a lysine residue. The IODOGEN method (Fraker er al (1978)
Biochem. Biophys. Res. Comm. 80, 49-57) can be used to incorporate
iodine-123. Reference ("Monoclonal Antibodies in
Immunoscintigraphy", J-F Chatal, CRC Press, 1989) describes other
methods in detail.
[0089] In a further preferred embodiment of the invention the
further moiety is able to bind selectively to a directly or
indirectly cytotoxic moiety or to a readily detectable moiety.
Thus, in this embodiment, the further moiety may be any moiety
which binds to a further compound or component which is cytotoxic
or readily detectable.
[0090] The further moiety may, therefore be an antibody which
selectively binds to the further compound or component, or it may
be some other binding moiety such as streptavidin or biotin or the
like. The following examples illustrate the types of molecules that
are included in the invention; other such molecules are readily
apparent from the teachings herein.
[0091] A bispecific antibody wherein one binding site comprises the
moiety which selectively binds to ECSM1 or ECSM4 and the second
binding site comprises a moiety which binds to, for example, an
enzyme which is able to convert a substantially non-toxic prodrug
to a cytotoxic drug.
[0092] A compound, such as an antibody which selectively binds to
ECSM1 or ECSM4, to which is bound biotin. Avidin or streptavidin
which has been labelled with a readily detectable label may be used
in conjunction with the biotin labelled antibody in a two-phase
imaging system wherein the biotin labelled antibody is first
localised to the target site in the patient, and then the labelled
avidin or streptavidin is administered to the patient. Bispecific
antibodies and biotin/streptavidin (avidin) systems are reviewed by
Rosebrough (1996) Q J Nucl. Med. 40, 234-251.
[0093] In a preferred embodiment of the invention, the moiety which
selectively binds to ECSM1 or ECSM4 and the further moiety are
polypeptides which are fused.
[0094] The compounds of the first and second aspects of the
invention are useful in treating, imaging or diagnosing disease,
particularly diseases in which there may be undesirable
neovasculature formation, as described in more detail below.
[0095] In a preferred embodiment of the first and second aspects of
the invention, the compounds are suitable for use in medicine.
[0096] A third aspect of the invention provides a nucleic acid
molecule encoding a compound of either the first or second aspects
of the invention wherein the selective binding moiety and the
further moiety are polypeptides which are fused.
Methods of Linking Polynucleotides are Described in More Detail
Below.
[0097] A fourth aspect of the invention provides a pharmaceutical
composition comprising a compound according to the invention and a
pharmaceutically acceptable carrier. The compound of the invention
includes those described in the first, second and third aspects.
The invention also includes pharmaceutical composition comprising
any of an antibody, polypeptide, peptide, polynucleotide,
expression vector or other agent which may be delivered to an
individual as described below and a pharmaceutically acceptable
carrier.
[0098] By "pharmaceutically acceptable" is included that the
formulation is sterile and pyrogen free. Suitable pharmaceutical
carriers are well known in the art of pharmacy.
[0099] The carrier(s) must be "acceptable" in the sense of being
compatible with the compound of the invention and not deleterious
to the recipients thereof. Typically, the carriers will be water or
saline which will be sterile and pyrogen free; however, other
acceptable carriers may be used.
[0100] Typically the pharmaceutical compositions or formulations of
the invention are for parenteral administration, more particularly
for intravenous administration.
[0101] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents.
[0102] A fifth aspect of the invention provides a method of imaging
vascular endothelium in the body of an individual the method
comprising administering to the individual an effective amount of a
compound according to either of the first or second aspects of the
invention wherein the further moiety is a readily detectable
moiety.
[0103] Typically the vascular endothelium is associated with
angiogenesis.
[0104] As discussed above in relation to the first and second
aspects of the invention, the moiety of the compound which
selectively binds ECSM4 or ECSM1 may be an antibody. Preferred
antibodies are as outlined above.
[0105] In a preferred embodiment of this aspect of the invention,
the method of imaging the vascular endothelium in an individual
comprises the further step of detecting the location of the
compound in the individual.
[0106] Detecting the compound or antibody can be achieved using
methods well known in the art of clinical imaging and diagnostics.
The specific method required will depend on the type of detectable
label attached to the compound or antibody. For example,
radioactive atoms may be detected using autoradiography or in some
cases by magnetic resonance imaging (MRI) as described above.
[0107] Imaging the vascular endothelium in the body is useful
because it can provide information about the health of the body. It
is particularly useful when the vascular endothelium is diseased,
or is proliferating due to a cancerous growth. Imaging cancer in a
patient is especially useful, because it can be used to determine
the size of a tumour and whether it is responding to treatment.
Since metastatic disease involves new blood vessel formation, the
method is useful in assessing whether metastasis has occurred.
[0108] Hence, in a preferred embodiment of the fifth aspect of the
invention, the vascular endothelium is neovasculature, such as that
produced in cancer.
[0109] A sixth aspect of the invention provides a method of
diagnosing or prognosing in an individual a condition which
involves the vascular endothelium the method comprising
administering to the individual an effective amount of a compound
according to either of the first or second aspects of the invention
wherein the further moiety is a readily detectable moiety.
[0110] The condition may be one which involves aberrant or
excessive growth of vascular endothelium, such as cancer,
artheresclerosis, restenosis, diabetic retinopathy, arthritis,
psoriasis, endometriosis, menorrhagia, haemangiomas and venous
malformations.
[0111] As discussed in relation to the first and second aspects of
the invention, the compound may comprise an antibody. The antibody
may be any antibody which selectively binds the polypeptide ECSM1
or ECSM4 as required. Preferred antibodies which bind the
polypeptide ECSM4 are as outlined above.
[0112] The method may be one which is an aid to diagnosis.
[0113] In a preferred embodiment of this aspect of the invention,
the method of diagnosing, or aiding diagnosis of, a condition
involving the vascular endothelium in an individual comprises the
further step of detecting the location of the compound in the
individual. Preferably the endothelium is in neovasculature; ie,
angiogenic vasculature.
[0114] The function of ECSM4 or ECSM1 may not be to promote
proliferation of vascular endothelial cells. Therefore the level of
expression of these polypeptides within an endothelial cell may not
be informative about the health of the vascular endothelium.
However, the location of expression of the polypeptides may be
informative, as they represent the growth of blood vessels.
Abnormal cell proliferation such as cancer may be diagnosed by the
detection of new vasculature.
[0115] A seventh aspect of the invention provides a method of
treating an individual in need of treatment, the method comprising
administering to the individual an effective amount of a compound
according to the first or second aspects of the invention wherein
the further moiety is a cytotoxic or therapeutic moiety.
[0116] In one embodiment of this aspect, the patient in need of
treatment has a proliferative disease or a condition involving the
vascular endothelium.
[0117] A number of diseases and conditions involve undesirable
neovasculature formation. Neovasculature formation is associated
with cancer, psoriasis, atherosclerosis, menorrhagia, arthritis
(both inflammatory and rheumatoid), macular degeneration, Paget's
disease, retinopathy and its vascular complications (including
proliferative and of prematurity, and diabetic), benign vascular
proliferations and fibroses.
[0118] By cancer is included Kaposi's sarcoma, leukaemia, lymphoma,
myeloma, solid carcinomas (both primary and secondary (metastasis),
vascular tumours including haemangioma (both capillary and juvenile
(infantile)), haemangiomatosis and haemagioblastoma.
[0119] Thus, the invention comprises a method of treating a patient
who has a disease in which angiogenesis contributes to pathology
the method comprising the step of administering to the patient an
effective amount of a compound of the first or second aspect of the
invention wherein the further moiety of the compound is one which
either directly or indirectly is of therapeutic benefit to the
patient.
[0120] Typically, the disease is associated with undesirable
neovasculature formation and the treatment reduces this to a useful
extent.
[0121] The tumours that may be treated by the methods of the
invention include any tumours which are associated with new blood
vessel production.
[0122] The term "tumour" is to be understood as referring to all
forms of neoplastic cell growth, including tumours of the lung,
liver, blood cells, skin, pancreas, stomach, colon, prostate,
uterus, breast, lymph glands and bladder. Solid tumours are
especially suitable. However, blood cancers, including leukaemias
and lymphomas are now also believed to involve new blood vessel
formation and may be treated by the methods of the invention.
[0123] Typically in the above-mentioned methods of treatment, the
further moiety is one which destroys or slows or reverses the
growth of the neovasculature.
[0124] It will readily be appreciated that, depending on the
particular compound used in imaging, diagnosis or treatment, the
timing of administration may vary and the number of other
components used in therapeutic systems disclosed herein may
vary.
[0125] For example, in the case where the compound of the invention
comprises a readily detectable moiety or a directly cytotoxic
moiety, it may be that only the compound, in a suitable
formulation, is administered to the patient. Of course, other
agents such as immunosuppressive agents and the like may be
administered.
[0126] In respect of compounds which are detectably labelled,
imaging takes place once the compound has localised at the target
site.
[0127] However, if the compound is one which requires a further
component in order to be useful for treatment, imaging or
diagnosis, the compound of the invention may be administered and
allowed to localise at the target site, and then the further
component administered at a suitable time thereafter.
[0128] For example, in respect of the ADEPT and ADEPT-like systems
above, the binding moiety-enzyme moiety compound is administered
and localises to the target site. Once this is done, the prodrug is
administered.
[0129] Similarly, for example, in respect of the compounds wherein
the further moiety comprised in the compound is one which binds a
further component, the compound may be administered first and
allowed to localise at the target site, and subsequently the
further component is administered.
[0130] Thus, in one embodiment a biotin-labelled anti-ECSM1 or
-ECSM4 antibody is administered to the patient and, after a
suitable period of time, detectably labelled streptavidin is
administered. Once the streptavidin has localised to the sites
where the antibody has localised (ie the target sites) imaging
takes place.
[0131] Where the compound whose moiety which selectively binds is
an antibody, the antibody may be any antibody which selectively
binds the polypeptide ECSM1 or ECSM4 as required. Preferred
antibodies are as outlined in the first and second aspects of the
invention.
[0132] It is believed that the compounds of the invention wherein
the further moiety is a readily detectable moiety may be useful in
determining the angiogenic status of tumours or other disease
states in which angiogenesis contributes to pathology. This may be
an important factor influencing the nature and outcome of future
therapy.
[0133] An eighth aspect of the invention provides a method of
introducing genetic material selectively into vascular endothelial
cells the method comprising contacting the cells with a compound
according to either of the first or second aspects of the invention
as described above wherein the further moiety is a nucleic
acid.
[0134] The vascular endothelial cells may be any vascular
endothelial cells such as those in tissue culture or in a living
organism. It is preferred if the cells are in a living organism. It
is further preferred if the organism is a human. It is still more
preferred if the vascular endothelial cells are those in
neovasculature, ie they are angiogenic endothelial cells.
[0135] Preferably, the binding moiety is an antibody. The antibody
may be any antibody which selectively binds the polypeptide ECSM1
or ECSM4 as required. Preferably, the antibody is one as defined
above in relation to the first or second aspects of the invention.
Typically, the binding moiety is comprised in a delivery vehicle
and preferably, the delivery vehicle is a liposome, as described in
further detail above. In this embodiment, the further moiety is
nucleic acid and is comprised within the liposome, also as
described above. Typically, the method is used in gene therapy, and
the genetic material is therapeutically useful. Therapeutically
useful genetic material includes that which encodes a therapeutic
protein.
[0136] A ninth aspect of the invention provides a use of a compound
according to either of the first or second aspects of the invention
wherein the further moiety is a readily detectable label in the
manufacture of a diagnostic or prognostic agent for a condition
which involves the vascular endothelium.
[0137] As discussed above, the compound may comprise an antibody as
the moiety which selectively binds. The antibody may be any
antibody which selectively binds the polypeptide ECSM1 or ECSM4 as
required.
[0138] A tenth aspect of the invention provides a use of a compound
according to either of the first or second aspects of the invention
wherein the further moiety is a cytotoxic or therapeutic moiety in
the manufacture of a medicament for treating a condition involving
the vascular endothelium.
[0139] Conditions which involve the vascular endothelium are
described above.
[0140] As described above, the compound may comprise an antibody as
the moiety which selectively binds. The antibody may be any
suitable antibody which selectively binds the polypeptide ECSM1 or
ECSM4 as required.
[0141] An eleventh aspect of the invention provides a polypeptide
comprising or consisting of a fragment or variant or fusion of the
ECSM4 polypeptide or a fusion of said fragment or variant provided
that it is not a polypeptide consisting of the amino acid sequence
given between residues 49 and 466 of FIG. 4.
[0142] The ECSM4 polypeptide includes a polypeptide comprising or
consisting of the amino acid sequence given in FIG. 4 or FIG. 5 or
FIG. 7 or FIG. 12 or FIG. 13 or the polypeptide encoded by the
nucleotide sequence of either FIG. 4 between positions 1 and 1395
or FIG. 5 between positions 2 and 948 or FIG. 7 or FIG. 12 or FIG.
13 is that of the ECSM4 polypeptide. Preferably, the ECSM4
polypeptide of the invention comprises but does not consist of the
amino acid sequence given in FIG. 4.
[0143] Preferably, the ECSM4 polypeptide of the invention does not
consist of any of the amino acid sequences represented by SEQ ID No
18085 of EP 1 074 617, SEQ ID No 211 of either WO 00/53756 or
WO99/46281, SEQ ID Nos 24-27, 29, 30, 33, 34, 38 or 39 of WO
01/23523, or SEQ ID No 86 of WO 99/11293, or any of the amino acid
sequences encoded by SEQ ID No 18084 or 5096 of EP 1 074 617, SEQ
ID No 210 of WO 00/53756 or WO 99/46281, or SEQ ID Nos 22, 23, 96
or 98 of WO 01/23523 or SEQ ID No 31 of WO 99/11293.
[0144] A twelfth aspect of the invention provides a polypeptide
comprising or consisting of the ECSM1 polypeptide or a fragment or
variant or fusion thereof or a fusion of said fragment or
variant.
[0145] The ECSM1 polypeptide includes a polypeptide comprising or
consisting of the amino acid sequence given in FIG. 2. Preferably,
the ECSM1 polypeptide or fragment is not a polypeptide whose
sequence is given in SEQ ID No 120 of WO 99/06423 or which is
encoded by SEQ ID No 32 of WO 99/06423 or encoded by the nucleic
acid of ATCC deposit No 209145 made on Jul. 17, 1997 for the
purposes of WO 99/06423.
[0146] The invention includes peptides which are derived from the
ECSM4 or ECSM1 polypeptides. These peptides may be considered
"fragments" of the ECSM4 or ECSM1 polypeptides but may be produced
by de novo synthesis or by fragmentation of the polypeptide.
[0147] "Fragments" of the ECSM4 or ECSM1 polypeptide include
polypeptides which comprise at least five consecutive amino acids
of the ECSM4 or ECSM1 polypeptide. Preferably, a fragment of the
polypeptide comprises an amino acid sequence which is useful, for
example, a fragment which retains activity of the polypeptide, or a
fragment for use in a binding assay or is useful as a peptide for
producing an antibody which is specific for the ECSM4 or ECSM1
polypeptide. An activity of the ECSM4 polypeptide may be in
endothelial cell repulsive guidance. Repulsive guidance may be
tested in vivo by constructing appropriate transgenic or knock-out
animal models, for example mice or zebrafish. It may also be tested
in vivo on cell migration assays such as Boyden chamber or video
microscopy. Typically, the fragments have at least 8 consecutive
amino acids, preferably at least 10, more preferably at least 12 or
15 or 20 or 30 or 40 or 50 consecutive amino acids of the ECSM4 or
ECSM1 polypeptide. Preferably, fragments of the ECSM4 polypeptide
comprise but do not consist of the amino acid sequence given in
FIG. 4 or FIG. 5 or FIG. 7 or FIG. 12 or FIG. 13. Preferably,
fragments of the ECSM4 polypeptide comprise but do not consist of
any of the amino acid sequences represented by SEQ ID No 18085 of
EP 1 074 617, SEQ ID No 211 of either WO 00/53756 or WO99/46281,
SEQ ID Nos 24-27, 29, 30, 33, 34, 38 or 39 of WO 01/23523, or SEQ
ID No 86 of WO 99/11293, or any of the amino acid sequences encoded
by SEQ ID No 18084 or 5096 of EP 1 074 617, SEQ ID No 210 of WO 00
53756 or WO 99/46281, or SEQ ID Nos 22, 23, 96 or 98 of WO 01/23523
or SEQ ID No 31 of WO 99/11293.
[0148] Typically, the fragments of ECSM4 polypeptide are ones which
have portions of the amino acid sequence shown in FIG. 4 or FIG.
12.
[0149] Typically, the fragments of ECSM1 polypeptide are ones which
have portions of the amino acid sequence shown in FIG. 2.
[0150] In a preferred embodiment of the thirteenth aspect of the
invention, a fragment of the ECSM4 polypeptide is a fragment which
has the sequence LSQSPGAVPQALVAWRA, DSVLTPEEVALCLEL, TYGYISVPTA,
KGGVLLCPPRPCLTPT, WLADTW, WLADTWRSTSGSRD, SPPTTYGYIS,
GSLANGWGSASEDNAASARASLVSSSDGSFLAD or FARALAVAVD or has a sequence
of at least 5 or 8 or 10 residues of any of these sequences. These
peptides correspond to amino acids 165-181, 274-288, 311-320,
336-351, 8-13, 8-21, 307-316, 355-387 and 390-399 respectively of
the human ECSM4 polypeptide shown in FIG. 4. Peptides WLADTW,
WLADTWRSTSGSRD, SPPTTYGYIS, GSLANGWGSASEDNAASARASLVSSSDGSFLAD and
FARALAVAVD represent conserved regions between the mouse and human
homologues of the ECSM4 polypeptide, and between the ECSM4
polypeptide and the mouse dutt1 protein. The peptides
LSQSPGAVPQALVAWRA, DSVLTPEEVALCLEL, TYGYISVPTA and KGGVLLCPPRPCLTPT
may be useful in raising antibodies.
[0151] Preferred peptides are peptides of at least 5 or 8 or 10 or
12 or 15 or 20 consecutive amino acid residues from these conserved
sequences. Peptides of ECSM4 which affect cell migration and/or
growth and/or vascular development are particularly preferred. They
can be identified in suitable screening systems.
[0152] In a further preferred embodiment of this aspect of the
invention, a fragment of the ECSM4 polypeptide is a fragment which
has the sequence GGDSLLGGRGSL, LLQPPARGHAHDGQALSTDL, EPQDYTEPVE,
TAPGGQGAPWAEE or ERATQEPSEHGP or has a sequence of at least 5 or 8
or 10 residues of any of these sequences. These peptides correspond
to regions of the human ECSM4 polypeptide (located at residues
4-16, 91-109, 227-236, 288-300 and 444-455 respectively in the
sequence given in FIG. 12) which are not, or are poorly, conserved
in the mouse homologue (see FIG. 14). As described below, such
peptides may be particularly useful in raising antibodies to the
human ECSM4 polypeptide.
[0153] According to the transmembrane domain predicting software
program called PRED-TMR (available at the internet site
http://www.biophys.biol.uoa.gr) and an amino acid sequence
alignment with the human protein Robo1 (whose transmembrane region
is known), residues 1-467 as shown in FIG. 12 are likely to be
extracellular, and in addition to being extracellularly exposed,
may include the binding site of the natural ligand. Hence fragments
of ECSM4 which include or consist of a sequence within the
extracellular domain of residues 1-467 of FIG. 12 may represent
useful fragments for raising antibodies selective for cells
expressing ECSM4 on their surface and which may also be useful in
modulating the activity of the polypeptide ECSM4.
[0154] Hence, preferred fragments of the ECSM4 polypeptide are
those fragments of the polypeptide sequence of FIG. 12 which
comprise at least 1, 3 or 5, amino acid residues which are not
conserved when compared to the mouse ECSM4 (as shown in FIG. 13).
More preferably at least 7, 9, 11 or 13 amino acid residues in the
fragment are not conserved between human ECSM4 and mouse ECSM4, and
still more preferably at least 15, 17, 19 or 21 residues of the
fragment are not conserved between human ECSM4 and mouse ECSM4. The
sequence of such fragments may be determined from the alignment of
the human and mouse amino acid sequences shown in FIG. 14.
[0155] It will be appreciated that fragments of the ECSM4 or ECSM1
polypeptide of the invention are particularly useful when fused to
other polypeptides, such as glutathione-S-transferase (GST), green
fluorescent protein (GFP), vesicular stomatitis virus glycoprotein
(VSVG) or keyhole limpet haemacyanin (KLH). Fusions of the
polypeptide, or fusions of fragments or variants of the polypeptide
of the invention are included in the scope of the invention.
[0156] Other useful fragments of ECSM4 are those which are able to
bind a ligand selective for ECSM4. Suitable methods for
identification of ligands such as peptides or other molecules which
bind ECSM4 is discussed in more detail above. Such peptides or
other ECSM4-binding molecules can be used to identify the amino
acid sequences present in ECSM4 which are responsible for ligand
binding. Identification of those fragments of ECSM4 which, when
isolated from the rest of the molecule, are still able to bind a
ligand of ECSM4 can be achieved by means of a screen. Typically,
such a screen will comprise contacting a ligand of ECSM4 with a
test fragment of the ECSM4 polypeptide and determining if the test
fragment binds the ligand. Fragments of ECSM4 are within the scope
of the invention, and may be particularly useful in medicine. A
fragment of ECSM4 which binds the natural ECSM4 ligand may
neutralise the effect of the ligand and thereby affect endothelial
cell migration, growth and/or vascular development. Hence,
administration of fragments of ECSM4 may be useful in the treatment
of diseases or conditions where endothelial cell migration, growth
and/or vascular development need to be modulated. Examples of such
diseases include cancer and artherosclerosis.
[0157] A "fusion" of the ECSM4 or ECSM1 polypeptide or a fragment
or variant thereof provides a molecule comprising a polypeptide of
the invention and a further portion. It is preferred that the said
further portion confers a desirable feature on the said molecule;
for example, the portion may useful in detecting or isolating the
molecule, or promoting cellular uptake of the molecule. The portion
may be, for example, a biotin moiety, a radioactive moiety, a
fluorescent moiety, for example a small fluorophore or a green
fluorescent protein (GFP) fluorophore, as well known to those
skilled in the art. The moiety may be an immunogenic tag, for
example a Myc tag, as known to those skilled in the art or may be a
lipophilic molecule or polypeptide domain that is capable of
promoting cellular uptake of the molecule or the interacting
polypeptide, as known to those skilled in the art.
[0158] A "variant" of the ECSM4 or ECSM1 polypeptide includes
natural variants, including allelic variants and
naturally-occurring mutant forms and variants with insertions,
deletions and substitutions, either conservative or
non-conservative, where such changes do not substantially alter the
activity of the said polypeptide. In the case of the ECSM4
polypeptide, as an endothelial specific homologue of the human
roundabout 1 it may well be involved in endothelial cell repulsive
guidance. In addition, polypeptides which are elongated as a result
of an insertion or which are truncated due to deletion of a region
are included in the scope of the invention. For example, deletion
of cytoplasmically-located regions may be useful in creation of
"dominant negative" or "dominant positive" forms of the
polypeptide. Similarly, deletion of a transmembrane region of the
polypeptide may produce such forms.
[0159] By "conservative substitution" is intended combinations such
as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg;
and Phe, Tyr.
[0160] By "non-conservative substitution" we include other
substitutions, such as those where the substituted residue mimics a
particular modification of the replaced residue, for example a
phosphorylated tyrosine or serine may be replaced by aspartate or
glutamate due to the similarity of the aspartate or glutamate side
chain to a phosphorylated residue (ie they carry a negative charge
at neutral pH).
[0161] Further non-conservative substitutions which are included in
the term "variants" are point mutations which alter one, sometimes
two, and usually no more than three amino acids. Such mutations are
well known in the art of biochemistry and are usually designed to
insert or remove a defined characteristic of the polypeptide.
Another type of non-conservative mutation is the alteration or
addition of a residue to a cysteine or lysine residue which can
then be used with maleimide or succinimide cross-linking reagents
to covalently conjugate the polypeptide to another moiety.
Non-glycosylated proteins may be mutated to convert an asparagine
to the recognition motif N-X-S/T for N-linked glycosylation. Such a
modification may be useful to create a tag for purification of the
polypeptide using Concanavalin A-linked beads.
[0162] Such variants may be made using the methods of protein
engineering and site-directed mutagenesis well known in the
art.
[0163] Variants of the ECSM4 polypeptide include polypeptides
comprising a sequence with at least 65% identity to the amino acid
sequence given in FIG. 4 or FIG. 7 or FIG. 12 or FIG. 13,
preferably at least 70% or 80% or 85% or 90% identity to said
sequence, and more preferably at least 95% or 98% identity to said
amino acid sequence.
[0164] Variants of the ECSM1 polypeptide include polypeptides
comprising a sequence with at least 65% identity to the amino acid
sequence given in FIG. 2, preferably at least 70% or 80% or 85% or
90% identity to said sequence, and more preferably at least 95% or
98% identity to said amino acid sequence.
[0165] Percent identity can be determined by, for example, the
LALIGN program (Huang and Miller, Adv. Appl. Math. (1991)
12:337-357) at the Expasy facility site
(http://www.ch.embnet.org/software/LALIGN_form.html) using as
parameters the global alignment option, scoring matrix BLOSUM62,
opening gap penalty -14, extending gap penalty -4.
[0166] A thirteenth aspect of the invention provides a
polynucleotide encoding the ECSM4 polypeptide of the invention, or
the complement thereof or a polynucleotide which selectively
hybridises to either of these which polynucleotide is not any one
of the clones corresponding to GenBank Accession No AK000805 or the
ESTs whose GenBank Accession Nos are given in Table 11 or Table
12.
[0167] GenBank Accession No AK000805 correspond to a cDNA sequence
cloned in the vector pME18SFL3. ESTs listed in Table 11 represent
nucleotide sequences which can be assembled into the contig
sequence shown in FIG. 5. ESTs listed in Table 12 represent
nucleotide sequences which can be assembled into the mouse
nucleotide cluster sequence (Mm.27782) given in FIG. 7.
[0168] Preferably, the polynucleotide of this aspect of the
invention does not consist of any one of the nucleotide sequences
represented by SEQ ID No 18084 or 5096 of EP 1 074 617, SEQ ID No
210 of WO 00 53756 or WO 99/46281, or SEQ ID Nos 22, 23, 96 or 98
of WO 01/23523 or SEQ ID No 31 of WO 99/11293, or their
complement.
[0169] Also preferably, the polynucleotide of this aspect of the
invention is not a polynucleotide which encodes a polypeptide
consisting of the amino acid sequence represented by any one of SEQ
ID No 18085 of EP 1 074 617, SEQ ID No 211 of either WO 00/53756 or
WO99/46281, SEQ ID Nos 24-27, 29, 30, 33, 34, 38 or 39 of WO
01/23523, or SEQ ID No 86 of WO 99/11293
[0170] Polynucleotides of the thirteenth aspect of the invention
are described in more detail below.
[0171] A fourteenth aspect of the invention provides a
polynucleotide encoding the ECSM1 polypeptide or the complement
thereof or a polynucleotide which selectively hybridises to either
of these, according to the twelfth aspect of the invention provided
that the polynucleotide is not one present in ATCC deposit No
209145 or the clone corresponding to GenBank Accession No AC011526
or the ESTs whose GenBank Accession Nos are given in Table 10.
[0172] By "encoding a polypeptide according to the twelfth aspect
of the invention" we mean that the polynucleotide is one which
encodes an ECSM1 polypeptide of the invention and is not one which
encodes a polypeptide whose sequence is given in SEQ ID No 120 of
WO 99/06423 or which is encoded by SEQ ID No 32 or by the nucleic
acid included in the microbiological deposit corresponding to
American Type Culture Collection (ATCC) No. 209145 made on 17 Jul.
1997.
[0173] ATCC deposit No 209145 comprises a pSport1 vector which
includes a 765 base nucleotide sequence.
[0174] The polynucleotide sequence given in SEQ ID No 32 of WO
99/06423 is similar to the nucleotide sequence shown in FIG. 2. The
sequence of SEQ ID No 32 given in WO 99/06423 may be capable of
encoding part of the ECSM1 polypeptide of the invention. Due to
degeneracy of the genetic code however, a polynucleotide sequence
may encode the ECSM1 polypeptide of the invention without having a
nucleotide sequence as given in WO 99/06423. In a similar manner, a
polynucleotide sequence may encode the (full length) ECSM4
polypeptide of the invention without having the same sequence as
that given in FIG. 4 or FIG. 5 or FIG. 12. Such polynucleotides are
within the scope of this invention.
[0175] Hence, it will be appreciated that a polynucleotide of the
thirteenth aspect of the invention is preferably not one whose
nucleotide sequence is given in FIG. 4, and that a polynucleotide
of the fourteenth aspect of the invention is preferably not a
polynucleotide which is disclosed in WO 99/06423, such as SEQ ID No
32 disclosed therein or its complement or variants or the
corresponding cDNA sequence deposited under Accession No 209145 at
the ATCC or a polynucleotide fragment capable of encoding a
polypeptide whose amino acid sequence comprises the sequence given
in SEQ ID No 120 of WO 99/06423.
[0176] A polynucleotide of the thirteenth or fourteenth aspects of
the invention may encode a variant of the ECSM4 or ECSM1
polypeptide as described above. In addition, the insertions and/or
deletions within the ECSM4 or ECSM1 polypeptide may lead to
frameshift mutations which may encode truncated (or elongated)
polypeptide products, and insertions, deletions or other mutations
may lead to the introduction of stop codons which encode truncate
polypeptide products.
[0177] The polynucleotide of the invention may be DNA or RNA. It is
preferred if it is DNA.
[0178] The polynucleotide may or may not contain introns. It is
preferred if it does not contain introns.
[0179] The polynucleotide may be single stranded or double stranded
or a mixture of either.
[0180] The polynucleotide of the invention has at least 10
nucleotides, and preferably at least 15 nucleotides and more
preferably at least 30 nucleotides. In a further preference, the
polynucleotide is more than 50 nucleotides, more preferably at
least 100 nucleotides, and still more preferably the polynucleotide
is at least 500 nucleotides. The polynucleotide may be more than 1
kb, and may comprise more than 5 kb.
[0181] The invention also includes a polynucleotide which is able
to selectively hybridise to a polynucleotide which encodes the
ECSM4 or ECSM1 polypeptide or a fragment or variant or fusion
thereof, or a fusion of said variant or fragment. Preferably, said
polynucleotide is at least 10 nucleotides, more preferably at least
15 nucleotides and still more preferably at least 30 nucleotides in
length. The said polynucleotide may be longer than 100 nucleotides
and may be longer than 200 nucleotides, but preferably the said
polynucleotide is not longer than 250 nucleotides. Such
polynucleotides are useful in procedures as a detection tool to
demonstrate the presence of the polynucleotide in a sample. Such a
sample may be a sample of DNA, such as a bacterial colony, fixed on
a membrane or filter.
[0182] Preferably, the polynucleotide which is capable of
selectively hybridising as said is not any one of the nucleotide
sequences represented by SEQ ID No 18084 or 5096 of EP 1 074 617,
SEQ ID No 210 of WO 00 53756 or WO 99/46281, or SEQ ID Nos 22, 23,
96 or 98 of WO 01/23523 or SEQ ID No 31 of WO 99/11293.
[0183] By "selectively hybridise" we mean that the polynucleotide
hybridises under conditions of high stringency. DNA-DNA, DNA-RNA
and RNA-RNA hybridisation may be performed in aqueous solution
containing between 0.1.times.SSC and 6.times.SSC and at
temperatures of between 55.degree. C. and 70.degree. C. It is well
known in the art that the higher the temperature or the lower the
SSC concentration the more stringent the hybridisation conditions.
By "high stringency" we mean 2.times.SSC and 65.degree. C.
1.times.SSC is 0.15M NaCl/0.015M sodium citrate. Polynucleotides
which hybridise at high stringency are included within the scope of
the claimed invention.
[0184] In another embodiment, the polynucleotide can be used as a
primer in the polymerase chain reaction (PCR), and in this capacity
a polynucleotide of between 15 and 30 nucleotides is preferred. A
polynucleotide of between 20 and 100 nucleotides is preferred when
the fragment is to be used as a mutagenic PCR primer. It is
particularly preferred if the PCR primer (when not being used to
mutate a nucleic acid) contains about 15 to 30 contiguous
nucleotides (ie perfect matches) from the nucleotide sequence given
in FIG. 4 or FIG. 7 or FIG. 12 or FIG. 13 from the nucleotide
sequence given in FIG. 2. Clearly, if the PCR primers are used for
mutagenesis, differences compared to the sequence will be
present.
[0185] Primers which are suitable for use in a polymerase chain
reaction (PCR; Saiki et al (1988) Science 239, 487-491) are
preferred. Suitable PCR primers may have the following
properties:
[0186] It is well known that the sequence at the 5' end of the
oligonucleotide need not match the target sequence to be
amplified.
[0187] It is usual that the PCR primers do not contain any
complementary structures with each other longer than 2 bases,
especially at their 3' ends, as this feature may promote the
formation of an artifactual product called "primer dimer". When the
3' ends of the two primers hybridize, they form a "primed template"
complex, and primer extension results in a short duplex product
called "primer dimer".
[0188] Internal secondary structure should be avoided in primers.
For symmetric PCR, a 40-60% G+C content is often recommended for
both primers, with no long stretches of any one base. The classical
melting temperature calculations used in conjunction with DNA probe
hybridization studies often predict that a given primer should
anneal at a specific temperature or that the 72.degree. C.
extension temperature will dissociate the primer/template hybrid
prematurely. In practice, the hybrids are more effective in the PCR
process than generally predicted by simple T.sub.m
calculations.
[0189] Optimum annealing temperatures may be determined empirically
and may be higher than predicted. Taq DNA polymerase does have
activity in the 37-55.degree. C. region, so primer extension will
occur during the annealing step and the hybrid will be stabilised.
The concentrations of the primers are equal in conventional
(symmetric) PCR and, typically, within 0.1- to 1 nM range.
[0190] When a pair of suitable nucleic acids of the invention are
used in a PCR it is convenient to detect the product by gel
electrophoresis and ethidium bromide staining. As an alternative to
detecting the product of DNA amplification using agarose gel
electrophoresis and ethidium bromide staining of the DNA, it is
convenient to use a labelled oligonucleotide capable of hybridising
to the amplified DNA as a probe. When the amplification is by a PCR
the oligonucleotide probe hybridises to the interprimer sequence as
defined by the two primers. The probe may be labelled with a
radionuclide such as .sup.32P, .sup.33P and .sup.35S using standard
techniques, or may be labelled with a fluorescent dye. When the
oligonucleotide probe is fluorescently labelled, the amplified DNA
product may be detected in solution (see for example Balaguer et al
(1991) "Quantification of DNA sequences obtained by polymerase
chain reaction using a bioluminescence adsorbent" Anal. Biochem.
195, 105-110 and Dilesare et al (1993) "A high-sensitivity
electrochemiluminescence-based detection system for automated PCR
product quantitation" BioTechniques 15, 152-157.
[0191] PCR products can also be detected using a probe which may
have a fluorophore-quencher pair or may be attached to a solid
support or may have a biotin tag or they may be detected using a
combination of a capture probe and a detector probe.
[0192] Fluorophore-quencher pairs are particularly suited to
quantitative measurements of PCR reactions (eg RT-PCR).
Fluorescence polarisation using a suitable probe may also be used
to detect PCR products.
[0193] Oligonucleotide primers can be synthesised using methods
well known in the art, for example using solid-phase
phosphoramidite chemistry.
[0194] A polynucleotide or oligonucleotide primer of the invention
may contain one or more modified bases or may contain a backbone
which has been modified for stability purposes or for other
reasons. By modified we included for example, tritylated bases and
unusual bases such as inosine. A variety of modifications can be
made to DNA and RNA and these are included in the scope of the
invention.
[0195] In a preferred embodiment, the polynucleotides of the
invention are detectably labelled. Suitable detectable labels are
described in detail above.
[0196] A fifteenth aspect of the invention provides an expression
vector comprising a polynucleotide as described above. Typically,
the polynucleotides are those which encode the polypeptides ECSM1
or ECSM4 or a fragment, variant or fusion thereof.
[0197] By "expression vector" we mean one which is capable, in an
appropriate host, of expressing a polypeptide encoded by the
polynucleotide.
[0198] Such vectors may be useful in expressing the encoded
polypeptide in a host cell for production of useful quantities of
the polypeptide, or may be useful in medicine. Expression vectors
comprising a polynucleotide according to the thirteenth or
fourteenth aspects of the invention which are suitable for use in
gene therapy are within the scope of the invention. Administration
of a gene therapy vector capable of expressing the ECSM4
polypeptide may be useful in modulating or inhibiting angiogenesis,
since his polypeptide is likely to be a repulsive guidance
receptor. Similarly, gene therapy vectors capable of expressing
fragments or mutants of ECSM4 on the cell surface, which fragments
or mutants are capable of binding the ECSM4 cognate ligand but are
not able to convey the normal downstream signal (for example,
because the necessary cytosolic portion of the polypeptide is
deleted or mutated so as to not be functional or capable of binding
normally interacting cellular proteins) may also be useful in
modulating angiogenesis in an individual.
[0199] Hence, in a preferred embodiment, the vector is one which is
suitable for use in gene therapy. Examples of suitable vectors and
methods of their introduction into cells are given in more detail
below. In particular, the gene therapy methods and vectors
described in relation to the use of promoters of ECSM4 may also be
used in relation to the use of ECSM4 coding sequences or antisense
in gene therapy.
[0200] It will be appreciated that the polynucleotide comprised
within the expression vector of this aspect of the invention may be
one which encodes the polypeptide ECSM4 or ECSM1 or a fragment or
variant thereof, or the polynucleotide may be one which is capable
of selectively hybridising to the ECSM4 or ECSM1 coding region.
Polynucleotides which are capable of hybridising to the ECSM4 or
ECSM1 coding region are useful as antisense polynucleotides which
may decrease the expression level of ECSM4 or ECSM1 within a target
cell. The design of suitable and effective antisense
polynucleotides based on a known coding sequence is known in the
art of gene therapy.
[0201] Preferably, the expression vector of this aspect of the
invention is one which does not contain a polynucleotide sequence
represented by any one of SEQ ID No 18085 of EP 1 074 617, SEQ ID
No 211 of either WO 00/53756 or WO99/46281, SEQ ID Nos 24-27, 29,
30, 33, 34, 38 or 39 of WO 01/23523, or SEQ ID No 86 of WO 99/11293
or their complement. Also preferably, the said vector is one which
does not contain a polynucleotide encoding a polypeptide whose
amino acid sequence is represented by any one of SEQ ID No 18085 of
EP 1 074 617, SEQ ID No 211 of either WO 00/53756 or WO99/46281,
SEQ ID Nos 24-27, 29, 30, 33, 34, 38 or 39 of WO 01/23523, or SEQ
ID No 86 of WO 99/11293.
[0202] Both the amount of therapeutic protein or therapeutic
polynucleotide produced and the duration of production are
important issues in gene therapy. Consequently, the use of viral
vectors capable of cellular gene integration (eg retroviral
vectors) may be more beneficial than non-integrating alternatives
(eg adenovirus derived vectors) when repeated therapy is
undesirable for immunogenicity reasons.
[0203] By "therapeutic polynucleotide" or "therapeutic protein" we
include ECSM4 and ECSM1 coding sequences, the polypeptide product
encoded by said coding sequences, and ECSM4 antisense
polynucleotides. The therapeutic effect of said polynucleotides or
proteins may include pro-angiogenic or anti-angiogenic effects,
depending on the precise therapeutic agent administered. For
example, an expression vector suitable for gene therapy which
comprises a polynucleotide which is antisense to at least part of
the ECSM4 coding region may have anti-angiogenic activity when
expressed in a host cell or patient if it suppresses expression of
a molecule which is required for angiogenesis. If the
polynucleotide comprised within the expression vector encodes a
polypeptide which is required for inhibition of angiogenesis (for
example, because said polypeptide has endothelial cell repulsive
guidance activity), then expression of the antisense may also be
anti-angiogenic.
[0204] Conversely, if said the expression vector comprises a
polynucleotide of the invention which polynucleotide suppresses
expression of a molecule whose activity is required to decrease
vascular growth (for example, because said molecule is an
endothelial cell repulsive guidance molecule) or encodes a
polypeptide whose activity is required for angiogenesis,
administration of the said vector may be pro-angiogenic.
[0205] Where the therapeutic gene is maintained extrachromosomally,
the highest level of expression is likely to be achieved using
viral promoters, for example, the Rous sarcoma virus long terminal
repeat (Ragot et al (1993) Nature 361, 647-650; Hyde et al (1993)
Nature 362, 250-255) and the adenovirus major late promoter. The
latter has been used successfully to drive the expression of a
cystic fibrosis transmembrane conductance regulator (CFTR) gene in
lung epithelium (Rosenfeld et al (1992) Cell 68, 143-155). Since
these promoters unction in a broad range of tissues they may not be
suitable to direct cell-type-specific expression unless the
delivery method can be adapted to provide the specificity. However,
somatic enhancer sequences could be used to give cell-type-specific
expression in an extrachromosomal setting.
[0206] As described in more detail below, the ECSM4
regulatory/promoter region is an example of a regulatory region
capable of conferring endothelial cell selective expression,
preferably selective to endothelial cells of neovasculature (ie,
angiogenic endothelial cells) on an operatively linked coding
region. As outlined above, such a coding region may encode an
antisense polynucleotide.
[0207] Where withdrawal of the gene-vector construct is not
possible, it may be necessary to add a suicide gene to the system
to abort toxic reactions rapidly. The herpes simplex virus
thymidine kinase gene, when transduced into cells, renders them
sensitive to the drug ganciclovir, creating the option of killing
the cells quickly.
[0208] The use of ectotropic viruses, which are species specific,
may provide a safer alternative to the use of amphotropic viruses
as vectors in gene therapy. In this approach, a human homologue of
the non-human, ectotropic viral receptor is modified in such a way
so as to allow recognition by the virus. The modified receptor is
then delivered to cells by constructing a molecule, the front end
of which is specified for the targeted cells and the tail part
being the altered receptor. Following delivery of the receptor to
its target, the genetically engineered ectotropic virus, carrying
the therapeutic gene, can be injected and will only integrate into
the targeted cells.
[0209] Virus-derived gene transfer vectors can be adapted to
recognise only specific cells so it may be possible to target to an
endothelial cell, such as endothelial cells within a tumour.
Similarly, it is possible to target expression of an therapeutic
gene to the endothelial cell, using an endothelial cell-specific
promoter such as that for the ECSM4 or ECSM1 genes.
[0210] One of the ECSM genes or a part of the genes or a
polynucleotide comprising an antisense to the gene may be
introduced into the cell in a vector such that the gene remains
extrachromosomal. In such a situation, the gene will be expressed
by the cell from the extrachromosomal location. Vectors for
introduction of genes both for recombination and for
extrachromosomal maintenance are known in the art, and any suitable
vector may be used. Methods for introducing DNA into cells such as
electroporation, calcium phosphate co-precipitation and viral
transduction are known in the art, and the choice of method is
within the competence of the ordinary skilled person. Cells
transformed with the wild-type novel gene can be used as model
systems to study cancer remission and drug treatments which promote
such remission.
[0211] A variety of methods have been developed to operably link
polynucleotides, especially DNA, to vectors, for example, via
complementary cohesive termini. For instance, complementary
homopolymer tracts can be added to the DNA segment to be inserted
into the vector DNA. The vector and DNA segment are then joined by
hydrogen bonding between the complementary homopolymeric tails to
form recombinant DNA molecules.
[0212] Synthetic linkers containing one or more restriction sites
provide an alternative method of joining the DNA segment to
vectors. The DNA segment, generated by endonuclease restriction
digestion as described earlier, is treated with bacteriophage T4
DNA polymerase or E. coli DNA polymerase I, enzymes that remove
protruding, 3'-single-stranded termini with their
3'-5'-exonucleolytic activities, and fill in recessed 3'-ends with
their polymerising activities.
[0213] The combination of these activities therefore generates
blunt-ended DNA segments. The blunt-ended segments are then
incubated with a larger molar excess of linker molecules in the
presence of an enzyme that is able to catalyse the ligation of
blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
Thus, the products of the reaction are DNA segments carrying
polymeric linker sequences at their ends. These DNA segments are
then cleaved with the appropriate restriction enzyme and ligated to
an expression vector that has been cleaved with an enzyme that
produces termini compatible with those of the DNA segment.
[0214] Synthetic linkers containing a variety of restriction
endonuclease site are commercially available from a number of
sources including International Biotechnologies Inc., New Haven,
Conn., USA.
[0215] A desirable way to modify the DNA encoding the polypeptide
of the invention is to use PCR. This method may be used for
introducing the DNA into a suitable vector, for example by
engineering in suitable restriction sites, or it may be used to
modify the DNA in other useful wasy as is known in the art.
[0216] In this method the DNA to be enzymatically amplified is
flanked by two specific primers which themselves become
incorporated into the amplified DNA. The said specific primers may
contain restriction endonuclease recognition sites which can be
used for cloning into expression vectors using methods known in the
art.
[0217] The DNA (or in the case of retroviral vectors, RNA) is then
expressed in a suitable host to produce a polypeptide comprising
the polypeptide of the invention. Thus, the DNA encoding the
polypeptide constituting the polypeptide of the invention may be
used in accordance with known techniques, appropriately modified in
view of the teachings contained herein, to construct an expression
vector, which is then used to transform an appropriate host cell
for the expression and production of the polypeptide of the
invention. Such techniques include those disclosed in U.S. Pat. No.
4,440,859 issued 3 Apr. 1984 to Rutter et al, U.S. Pat. No.
4,530,901 issued 23 Jul. 1985 to Weissman, U.S. Pat. No. 4,582,800
issued 15 Apr. 1986 to Crowl, U.S. Pat. No. 4,677,063 issued 30
Jun. 1987 to Mark et al, U.S. Pat. No. 4,678,751 issued 7 Jul. 1987
to Goeddel, U.S. Pat. No. 4,704,362 issued 3 Nov. 1987 to Itakura
et al, U.S. Pat. No. 4,710,463 issued 1 Dec. 1987 to Murray, U.S.
Pat. No. 4,757,006 issued 12 Jul. 1988 to Toole, Jr. et al, U.S.
Pat. No. 4,766,075 issued 23 Aug. 1988 to Goeddel et al and U.S.
Pat. No. 4,810,648 issued 7 Mar. 1989 to Stalker, all of which are
incorporated herein by reference.
[0218] The DNA (or in the case or retroviral vectors, RNA) encoding
the polypeptide constituting the polypeptide of the invention may
be joined to a wide variety of other DNA sequences for introduction
into an appropriate host. The companion DNA will depend upon the
nature of the host, the manner of the introduction of the DNA into
the host, and whether episomal maintenance or integration is
desired.
[0219] Generally, the DNA is inserted into an expression vector,
such as a plasmid, in proper orientation and correct reading frame
for expression. If necessary, the DNA may be linked to the
appropriate transcriptional and translational regulatory control
nucleotide sequences recognised by the desired host, although such
controls are generally available in the expression vector. The
vector is then introduced into the host through standard
techniques. Generally, not all of the hosts will be transformed by
the vector. Therefore, it will be necessary to select for
transformed host cells. One selection technique involves
incorporating into the expression vector a DNA sequence, with any
necessary control elements, that codes for a selectable trait in
the transformed cell, such as antibiotic resistance. Alternatively,
the gene for such selectable trait can be on another vector, which
is used to co-transform the desired host cell.
[0220] Host cells that have been transformed by the expression
vector of the invention are then cultured for a sufficient time and
under appropriate conditions known to those skilled in the art in
view of the teachings disclosed herein to permit the expression of
the polypeptide, which can then be recovered.
[0221] Many expression systems are known, including bacteria (for
example, E. coli and Bacillus subtilis), yeasts (for example
Saccharomyces cerevisiae), filamentous fungi (for example
Aspergillus), plant cells, animal cells and insect cells.
[0222] The vectors typically include a prokaryotic replicon, such
as the ColE1 ori, for propagation in a prokaryote, even if the
vector is to be used for expression in other, non-prokaryotic, cell
types. The vectors can also include an appropriate promoter such as
a prokaryotic promoter capable of directing the expression
(transcription and translation) of the genes in a bacterial host
cell, such as E. coli, transformed therewith.
[0223] A promoter is an expression control element formed by a DNA
sequence that permits binding of RNA polymerase and transcription
to occur. Promoter sequences compatible with exemplary bacterial
hosts are typically provided in plasmid vectors containing
convenient restriction sites for insertion of a DNA segment of the
present invention.
[0224] Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322
and pBR329 available from Biorad Laboratories, (Richmond, Calif.,
USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway,
N.J., USA.
[0225] A typical mammalian cell vector plasmid is pSVL available
from Pharmacia, Piscataway, N.J., USA. This vector uses the SV40
late promoter to drive expression of cloned genes, the highest
level of expression being found in T antigen-producing cells, such
as COS-1 cells.
[0226] An example of an inducible mammalian expression vector is
pMSG, also available from Pharmacia. This vector uses the
glucocorticoid-inducible promoter of the mouse mammary tumour virus
long terminal repeat to drive expression of the cloned gene.
[0227] Useful yeast plasmid vectors are pRS403-406 and pRS413-416
and are generally available from Stratagene Cloning Systems, La
Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and
pRS406 are Yeast Integrating plasmids (YIps) and incorporate the
yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids
pRS413-416 are Yeast Centromere plasmids (Ycps).
[0228] Other vectors and expression systems are well known in the
art for use with a variety of host cells.
[0229] A sixteenth aspect of the invention provides a recombinant
host cell comprising a polynucleotide or vector of the
invention.
[0230] The polynucleotide of the invention includes polynucleotides
encoding a compound of the third aspect of the invention (where
both the moiety which selectively binds and the further moiety are
polypeptides which are fused) or an ECSM4 or ECSM1 polypeptide of
the invention or a fragment or fusion or variant thereof as defined
above.
[0231] The host cell can be either prokaryotic or eukaryotic.
Bacterial cells are preferred prokaryotic host cells and typically
are a strain of E. coli such as, for example, the E. coli strains
DH5 available from Bethesda Research Laboratories Inc., Bethesda,
Md., USA, and RR1 available from the American Type Culture
Collection (ATCC) of Rockville, Md., USA (No. ATCC 31343).
Preferred eukaryotic host cells include yeast, insect and mammalian
cells, preferably vertebrate cells such as those from a mouse, rat,
monkey or human fibroblastic and kidney cell lines. Yeast host
cells include YPH499, YPH500 and YPH501 which are generally
available from Stratagene Cloning Systems, La Jolla, Calif. 92037,
USA. Preferred mammalian host cells include Chinese hamster ovary
(CHO) cells available from the ATCC as CRL 1658 and 293 cells which
are human embryonic kidney cells. Preferred insect cells are SD
cells which can be transfected with baculovirus expression
vectors.
[0232] Transformation of appropriate cell hosts with a DNA
construct of the present invention is accomplished by well known
methods that typically depend on the type of vector used. With
regard to transformation of prokaryotic host cells, see, for
example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and
Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation
of yeast cells is described in Sherman et al (1986) Methods In
Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y. The
method of Beggs (1978) Nature 275, 104-109 is also useful. With
regard to vertebrate cells, reagents useful in transfecting such
cells, for example calcium phosphate and DEAE-dextran or liposome
formulations, are available from Stratagene Cloning Systems, or
Life Technologies Inc., Gaithersburg, Md. 20877, USA.
[0233] Electroporation is also useful for transforming and/or
transfecting cells and is well known in the art for transforming
yeast cells, bacterial cells, insect cells and vertebrate
cells.
[0234] For example, many bacterial species may be transformed by
the methods described in Luchansky et al (1988) Mol. Microbiol. 2,
637-646 incorporated herein by reference. The greatest number of
transformants is consistently recovered following electroporation
of the DNA-cell mixture suspended in 2.5 PEB using 6250V per cm at
25 .mu.FD.
[0235] Methods for transformation of yeast by electroporation are
disclosed in Becker & Guarente (1990) Methods Enzymol. 194,
182.
[0236] Successfully transformed cells, ie cells that contain a DNA
construct of the present invention, can be identified by well-known
techniques. For example, cells resulting from the introduction of
an expression construct of the present invention can be grown to
produce the polypeptide of the invention. Cells can be harvested
and lysed and their DNA content examined for the presence of the
DNA using a method such as that described by Southern (1975) J.
Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208.
Alternatively, the presence of the protein in the supernatant can
be detected using antibodies as described below.
[0237] In addition to directly assaying for the presence of
recombinant DNA, successful transformation can be confirmed by well
known immunological methods when the recombinant DNA is capable of
directing the expression of the protein. For example, cells
successfully transformed with an expression vector produce proteins
displaying appropriate antigenicity.
[0238] Samples of cells suspected of being transformed are
harvested and assayed for the protein using suitable
antibodies.
[0239] The host cell may be a host cell within an animal body.
Thus, transgenic animals which express a polypeptide of the first
or third aspects of the invention by virtue of the presence of the
transgene are included. Preferably, the transgenic animal is a
rodent such as a mouse. Transgenic animals can be made using
methods well known in the art.
[0240] Polynucleotides encoding the polypeptide ECSM4 may be useful
in generating transgenic non-human mammals wherein the ECSM4 is
mutated in some way. For example, the mouse ECSM4 genomic coding
region may be mutated in a mouse so as to produce an ECSM4
polypeptide which is incapable of binding its natural ligand, or
incapable of correctly interacting with intracellular components.
Such a mutated ECSM4 polypeptide may produce a disease in the mouse
which is very similar to a disease involving abnormal
vascularisation in humans.
[0241] Hence, non-human mammals, especially rodents such as mice
and rats, are useful as models of diseases involving abnormal
vascularisation.
[0242] Alternatively, mammals lacking the ECSM4 gene ("knock-outs")
or lacking an ECSM4 genomic coding region which is capable of being
transcribed or of expressing the ECSM4 polypeptide, may be useful
in providing a means of generating antibodies selective for the
human ECSM4 polypeptide. Such mammals, especially mice, are likely
to be particularly useful since the high level of homology between
the human and mouse ECSM4 polypeptides may prevent human ECSM4
polypeptide from being antigenic in mice who do express the ECSM4
polypeptide.
[0243] A potentially more accurate animal model of diseases
involving abnormal vascularisation may be made by addition to the
genome of a transgenic animal as described above, or replacing the
genomic ECSM4 of an animal with, the gene for human ECSM4 which has
been mutated. Suitably, the human ECSM4 inserted will be under
control of an endothelial selective promoter and regulatory region.
Preferably, the promoter and regulatory regions are those of the
host animal ECSM4 gene. An animal who genome is modified in this
way will express the dysfunctional human ECSM4, and therefore will
be useful in testing the efficacy of drugs and antibodies in the
diagnosis, prognosis and treatment of diseases involving abnormal
vascularisation in humans.
[0244] Such knockout or transgenic mammals are within the scope of
the invention and antibodies generated using such mammals and
compounds comprising them are also included within the scope of the
invention.
[0245] A seventeenth aspect of the invention provides a method of
producing a polypeptide of the invention, the method comprising
expressing a polynucleotide as described above or culturing a host
cell as described herein.
[0246] It will be appreciated that in order to produce the ECSM1
polypeptide, the host cell may comprise a polynucleotide encoding a
polypeptide whose amino acid sequence includes the sequence given
in FIG. 2, and that in order to produce the ECSM4 polypeptide the
host cell may comprise a polynucleotide encoding the polypeptide
whose amino acid sequence is given in FIG. 4 or FIG. 7 or FIG. 12
and so on.
[0247] Preferably, the polynucleotide expressed does not consist of
any one of the nucleotide sequences represented by SEQ ID No 18084
or 5096 of EP 1 074 617, SEQ ID No 210 of WO 00/53756 or WO
99/46281, or SEQ ID Nos 22, 23, 96 or 98 of WO 01/23523 and SEQ ID
No 31 of WO 99/11293.
[0248] Also preferably, the polypeptide produced is not one with an
amino acid sequence consisting of the sequence represented by any
one of SEQ ID No 18085 of EP 1 074 617, SEQ ID No 211 of either WO
00/53756 or WO99/46281, SEQ ID Nos 24-27, 29, 30, 33, 34, 38 or 39
of WO 01/23523, or SEQ ID No 86 of WO 99/11293.
[0249] Methods of cultivating host cells and isolating recombinant
proteins are well known in the art. It will be appreciated that,
depending on the host cell, the ECSM1 or ECSM4 polypeptides
produced may differ from that which can be isolated from nature.
For example, certain host cells, such as yeast or bacterial cells,
either do not have, or have different, post-translational
modification systems which may result in the production of forms of
ECSM1 or ECSM4 which may be post-translationally modified in a
different way to ECSM1 or ECSM4 isolated from nature. In order to
obtain ECSM1 or ECSM4 which is post-translationally modified in a
different way to human ECSM1 or ECSM4 it is preferred if the host
cell is a non-human host cell; more preferably it is not a
mammalian cell.
[0250] It is preferred that the ECSM1 or ECSM4 polypeptide is
produced in a eukaryotic system, such as an insect cell.
[0251] According to a less preferred embodiment, the ECSM1 or ECSM4
polypeptide can be produced in vitro using a commercially available
in vitro translation system, such as rabbit reticulocyte lysate or
wheatgerm lysate (available from Promega). Preferably, the
translation system is rabbit reticulocyte lysate. Conveniently, the
translation system may be coupled to a transcription system, such
as the TNT transcription-translation system (Promega). This system
has the advantage of producing suitable mRNA transcript from an
encoding DNA polynucleotide in the same reaction as the
translation. Conveniently, where the expressed polypeptide
comprises one or more transmembrane domains, the translation system
can be supplemented with a source of endoplasmic reticulum-derived
membranes and folding chaperones, such as dog pancreatic
microsomes, to allow synthesis of the polypeptide in a native
conformation.
[0252] Preferably, the production method of this aspect of the
invention comprises a further step of isolating the ECSM1 or ECSM4
produced from the host cell or from the in vitro translation mix.
Preferably, the isolation employs an antibody which selectively
binds the expressed polypeptide of the invention.
[0253] It will be understood that the invention comprises the ECSM1
or ECSM4 polypeptides or the variants or fragments or fusions
thereof, or a fusion of said variants or fragments obtainable by
the methods herein disclosed, provided that the ECSM4 polypeptide
is not one which consists of the amino acid sequence given in FIG.
4. Preferably, the polypeptide is not one which consists of an
amino acid sequence represented by any one of SEQ ID No 18085 of EP
1 074 617, SEQ ID No 211 of either WO 00/53756 or WO99/46281, SEQ
ID Nos 24-27, 29, 30, 33, 34, 38 or 39 of WO 01/23523, or SEQ ID No
86 of WO 99/11293. Preferably, the ECSM1 polypeptide produced by
the methods herein disclosed is not one which is encoded by SEQ ID
No 32 of WO 99/06423 or encoded by the nucleic acid of ATCC deposit
No. 209145 made on Jul. 17, 1997 for the purposes of WO
99/06423.
[0254] An eighteenth aspect of the invention provides an antibody
capable of selectively binding to either ECSM4 or ECSM1 as defined
above.
[0255] Preferably, an antibody which selectively binds ECSM1 is not
one which binds a polypeptide encoded by SEQ ID No 32 of WO
99/06423 or encoded by the nucleic acid of ATCC deposit No 209145
made on Jul. 17, 1997 for the purposes of the international patent
application PCT/US98/15949.
[0256] Preferably, an antibody which selectively binds ECSM1 is one
which binds a polypeptide whose amino acid sequence comprises the
sequence given in FIG. 2 or a natural variant thereof but does not
comprise the amino acid sequence encoded by ATCC deposit No 209145
made on Jul. 17, 1997.
[0257] Preferably, an antibody which selectively binds ECSM4 is one
which binds a polypeptide whose amino acid sequence comprises the
sequence given in any one of FIGS. 4, 5, 7, 12 or 13 or a natural
variant thereof but does not bind the polypeptide represented by
any one of SEQ ID No 18085 of EP 1 074 617, SEQ ID No 211 of either
WO 00/53756 or WO99/46281, SEQ ID Nos 24-27, 29, 30, 33, 34, 38 or
39 of WO 01/23523, or SEQ ID No 86 of WO 99/11293, or encoded by
any one of the nucleotide sequences represented by SEQ ID No 18084
or 5096 of EP 1 074 617, SEQ ID No 210 of WO 00/53756 or WO
99/46281, or SEQ ID Nos 22, 23, 96 or 98 of WO 01/23523 and SEQ ID
No 31 of WO 99/11293.
[0258] By "selectively bind" we include antibodies which bind at
least 10-fold more strongly to a polypeptide of the invention (such
as ECSM4 or ECSM1) than to another polypeptide; preferably at least
50-fold more strongly and more preferably at least 100-fold more
strongly. Such antibodies may be made by methods well known in the
art using the information concerning the differences in amino acid
sequence of ECSM4 or ECSM1 and another polypeptide which is not a
polypeptide of the invention.
[0259] Antibodies which selectively bind ECSM4 may also modulate
the function of the ECSM4 polypeptide. Antibodies which mimic the
effect of binding of the cognate ligand by stimulating or
activating ECSM4, or which bind and thereby prevent subsequent
binding and activation or stimulation of ECSM4 by the cognate
ligand, and such function-modulating antibodies are included in the
scope of the invention. It will be appreciated that antibodies
which modulate the function are useful as a tool in research, for
example in studying the effects of ECSM4 stimulation or activation,
or downstream processes triggered by such stimulation. Such
antibodies are also useful in medicine, for example in modulating
angiogenesis in an individual. Specifically, modulation of
angiogenesis by administration of such an antibody may be useful in
the treatment of a disease in an individual where modulation of
angiogenesis would be beneficial, such as cancer.
[0260] The following peptides may be useful as immunogens in the
generation of antibodies, such as rabbit polyclonal sera:
LSQSPGAVPQALVAWRA, DSVLTPEEVALCLEL, TYGYISVPTA and
KGGVLLCPPRPCLTPT.
[0261] In a preferred embodiment of this aspect, the antibody of
the invention selectively binds an amino acid sequence with the
sequence GGDSLLGGRGSL, LLQPPARGHAHDGQALSTDL, EPQDYTEPVE,
TAPGGQGAPWAEE or ERATQEPSEHGP. These sequences represent amino acid
sequences which are not identical between the human and mouse ECSM4
polypeptide sequences. Generally, the human and mouse ECSM4
polypeptides display a high degree of identity, which makes the
production of mouse antibodies to the human ECSM4 particularly
difficult due to the lack of immunogenicity of much of the human
ECSM4 sequence in mouse. Amino acid sequences which are absent from
the mouse ECSM4 are more likely to more be immunogenic in a mouse
than those sequences which are present in the mouse ECSM4 (an
alignment of the human and mouse ECSM4 amino acid sequences is
shown in FIG. 14). Hence, polypeptide fragments which contain
sequences which are unique to human ECSM4 as described above are
more useful than ECSM4 polypeptides whose sequence is found in both
human and mouse ECSM4, in the production of antibodies which
selectively bind the human ECSM4 polypeptide.
[0262] Antibodies generated as a result of use of amino acid
sequences which are located in the extracellular portion of the
ECSM4 polypeptide are likely to be useful as endothelial cell
targeting molecules. Therefore, it is particularly preferred if the
antibody of the invention is raised to, and preferably selectively
binds, an amino acid sequence which is unique to the human ECSM4
polypeptide, which sequence is located towards the N-terminal end
of the polypeptide and is found in the extracellular portion
located between residues 1 and 467 of the amino acid sequence given
in FIG. 12. An example of an amino acid sequence which is suitable
for raising antibody molecules selective for the ECSM4
extracellular region is given in FIG. 12.
[0263] Although the amino acid sequences which are unique to the
human ECSM4 may be used to produce polyclonal antibodies, it is
preferred if they are used to produce monoclonal antibodies.
[0264] Peptides in which one or more of the amino acid residues are
chemically modified, before or after the peptide is synthesised,
may be used providing that the function of the peptide, namely the
production of specific antibodies in vivo, remains substantially
unchanged. Such modifications included forming salts with acids or
bases, especially physiologically acceptable organic or in organic
acids and bases, forming an ester or amid of a terminal carboxyl
group, and attaching amino acid protecting groups such as
N-t-butoxycarbonyl. Such modifications may protect the peptide from
in vivo metabolism. The peptides may be present as single copies or
as multiples, for example tandem repeats. Such tandem or multiple
repeats may be sufficiently antigenic themselves to obviate the use
of a carrier. It may be advantageous for the peptide to be formed
as a loop, with the N-terminal and C-terminal ends joined together,
or to add one or more Cys residues to an end to increase
antigenicity and/or to allow disulphide bonds to be formed. If the
peptide is covalently linked to a carrier, preferably a
polypeptide, then the arrangement is preferably such that the
peptide of the invention forms a loop.
[0265] According to current immunological theories, a carrier
function should be present in any immunogenic formulation in order
to stimulate, or enhance stimulation of, the immune system. It is
though that the best carriers embody (or, together with the
antigen, create) a T-cell epitope. The peptides may be associated,
for example by cross-linking, with a separate carrier, such as
serum albumins, myoglobins, bacterial toxoids and keyhole limpit
haemocyanin. More recently developed carriers which induce T-cell
help in the immune response include the hepatitis-B core antigen
(also called the nucleocapsid protein), presumed T-cell epitopes
such as Thr-Ala-Ser-Gly-Val-Ala-Glu-Thr-Thr-Asn-Cys,
.beta.-galactosidase and the 163-171 peptide of interleukin-1. The
latter compound may variously be regarded as a carrier or as an
adjuvant or as both. Alternatively, several copies of the same or
different peptides of the invention may be cross-linked to one
another; in this situation there is no separate carrier as such,
but a carrier function may be provided by such cross-linking.
Suitably cross-linking agents include those listed as such in the
Sigma and Pierce catalogues, for example glutaraldehyde,
carbodimide and succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate, the latter agent
exploiting the --SH group on the C-terminal cysteine residue (if
present).
[0266] If the peptide is prepared by expression of a suitable
nucleotide sequence in a suitable host, then it may be advantageous
to express the peptide as a fusion product with a peptide sequence
which acts as a carrier. Kabigen's "Ecosec" system is an example of
such an arrangement.
[0267] Peptides may be synthesised by the Fmoc-polyamide mode of
solid-phase peptide synthesis as disclosed by Lu et al (1981) J.
Org. Chem. 46, 3433 and references therein. Temporary N-amino group
protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc)
group. Repetitive cleavage of this highly base-labile protecting
group is effected using 20% piperidine in N,N-dimethylformamide.
Side-chain functionalities may be protected as their butyl ethers
(in the case of serine threonine and tyrosine), butyl esters (in
the case of glutamic acid and aspartic acid), butyloxycarbonyl
derivative (in the case of lysine and histidine), trityl derivative
(in the case of cysteine) and
4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case
of arginine). Where glutamine or asparagine are C-terminal
residues, use is made of the 4,4'-dimethoxybenzhydryl group for
protection of the side chain amido functionalities. The solid-phase
support is based on a polydimethyl-acrylamide polymer constituted
from the three monomers dimethylacrylamide (backbone-monomer),
bisacryloylethylene diamine (cross linker) and acryloylsarcosine
methyl ester (functionalising agent). The peptide-to-resin
cleavable linked agent used is the acid-labile
4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid
derivatives are added as their preformed symmetrical anhydride
derivatives with the exception of asparagine and glutamine, which
are added using a reversed
N,N-dicyclohexyl-carbodiimide/4-hydroxybenzotriazole mediated
coupling procedure. All coupling and deprotection reactions are
monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin
test procedures. Upon completion of synthesis, peptides are cleaved
from the resin support with concomitant removal of side-chain
protecting groups by treatment with 95% trifluoroacetic acid
containing a 50% scavenger mix. Scavengers commonly used are
ethanedithiol, phenol, anisole and water, the exact choice
depending on the constituent amino acids of the peptide being
synthesised. Trifluoroacetic acid is removed by evaporation in
vacuo, with subsequent trituration with diethyl ether affording the
crude peptide. Any scavengers present are removed by a simple
extraction procedure which on lyophilisation of the aqueous phase
affords the crude peptide free of scavengers. Reagents for peptide
synthesis are generally available from Calbiochem-Novabiochem (UK)
Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any
one, or a combination of, techniques such as size exclusion
chromatography, ion-exchange chromatography and principally)
reverse-phase high performance liquid chromatography. Analysis of
peptides may be carried out using thin layer chromatography,
reverse-phase high performance liquid chromatography, amino-acid
analysis after acid hydrolysis and by fast atom bombardment (FAB)
mass spectrometric analysis.
[0268] The peptide of the invention may be linked to other antigens
to provide a dual effect.
[0269] Included in the scope of the invention is a method of
producing an antibody according to this aspect of the
invention.
[0270] Antibodies can be raised in an animal by immunising with an
appropriate peptide. Appropriate peptides are described herein.
Alternatively, with today's technology, it is possible to make
antibodies as defined herein without the need to use animals. Such
techniques include, for example, antibody phage display technology
as is well known in the art. Appropriate peptides, as described
herein, may be used to select antibodies produced in this way.
[0271] It will be appreciated that, with the advancements in
antibody technology, it may not be necessary to immunise an animal
in order to produce an antibody. Synthetic systems, such as phage
display libraries, may be used. The use of such systems is included
in the methods of the invention and the products of such systems
are "antibodies" for the purposes of the invention.
[0272] It will be appreciated that such antibodies which recognise
ECSM1 or ECSM4 and variants or fragments thereof are useful
research reagents and therapeutic agents, particularly when
prepared as a compound of the invention as described above.
Suitably, the antibodies of the invention are detectably labelled,
for example they may be labelled in such a way that they may be
directly or indirectly detected. Conveniently, the antibodies are
labelled with a radioactive moiety or a coloured moiety or a
fluorescent moiety, or they may be linked to an enzyme. Typically,
the enzyme is one which can convert a non-coloured (or
non-fluorescent) substrate to a coloured (or fluorescent) product.
The antibody may be labelled by biotin (or streptavidin) and then
detected indirectly using streptavidin (or biotin) which has been
labelled with a radioactive moiety or a coloured moiety or a
fluorescent moiety, or the like or they may be linked to any enzyme
of the type described above.
[0273] A nineteenth aspect of the invention provides a method of
detecting endothelial damage or activation in an individual
comprising obtaining a fluid sample from the individual and
detecting the presence of fragments of ECSM1 or ECSM4 in the
sample.
[0274] Preferably, the fluid sample is blood. Typically, the
presence of peptide fragments derived from ECSM1 or ECSM4 are
detected.
[0275] In a preferred embodiment of this aspect, the presence of
peptide fragments of the ECSM1 or ECSM4 polypeptides are detected
using an antibody selective for a polypeptide whose amino acid
sequence comprises a sequence given in either one of FIG. 2 or FIG.
4 or FIG. 12 or fragments thereof. Preferably, the antibody is an
antibody according to the eighteenth aspect of the invention.
Typically, such an antibody would be detectably labelled.
[0276] Detecting or diagnosing endothelial cell damage in an
individual is useful in diagnosing cancer or aiding diagnosis of
cardiac disease, endometriosis or artheroslcerosis in that
individual. It may be that certain levels of apparent cell damage
are detected in individuals who do not have cancer, cardiac
disease, endometriosis or artheroslcerosis. It may be necessary to
compare the amount of endothelial cell damage detected with amounts
or levels observed in individuals who are known to have cancer,
cardiac disease, endometriosis or artheroslcerosis with the
"normal" levels of apparent damage in the individual who does not
have cancer, cardiac disease, endometriosis or
artheroslcerosis.
[0277] Hence, detection of endothelial damage or activation in an
individual may be useful as a means of detecting the presence or
extent or growth rate of a tumour in that individual. The detection
of vessel damage is an indirect report of the formation of tumour
neovasculature. In this way, ECSM4 or ECSM1 may be surrogate
markers of angiogenesis. The presence of ECSM4 or ECSM1 fragments
in a sample from the individual, or more ECSM4 or ECSM1 polypeptide
fragments than in an individual who does not have a tumour, may be
a means of detecting a tumour, or growth of a known tumour, in that
individual.
[0278] Furthermore, it will be appreciated that detection of
neovasculature by means of detecting the presence of, or a certain
level of, ECSM4 or ECSM1 in a sample from an individual may be
useful in determining if a treatment in that individual is being
effective, and/or to what extent the treatment is effective.
Preferably the therapy is to treat a tumour or cancer in the
individual.
[0279] Hence, an aspect of the invention provides a method of
detecting a tumour or tumour neovasculature or cardiac disease or
endometriosis or artherosclerosis in an individual comprising
obtaining a fluid sample from the individual and detecting the
presence of fragments of ECSM1 or ECSM4 in the sample.
[0280] As described above in relation to detecting or diagnosing
endothelial cell damage, detection of the disease (such as 4 tumour
or cardiac disease etc) by means of detecting the presence of, or a
certain level of, ECSM4 or ECSM1 in a sample from an individual may
be useful in determining the efficacy of a treatment in that
individual.
[0281] In one embodiment, the therapy is gene therapy.
[0282] Preferably, the efficacy of the a treatment in an individual
is determined using the amount of fragments of ECSM1 or ECSM4 found
in the fluid sample of the individual and comparing it to either to
the amount of ECSM1 or ECSM4 fragments in a sample from an
individual who does not have cancer, cardiac disease, endometriosis
or artherosclerosis and/or to the amount in a sample from the
individual prior to commencement of said treatment. The comparison
indicates the efficacy of treatment of the individual, wherein if
there is no change in the amount of fragments determined before and
during/after treatment this is indicative of poor efficacy of the
treatment. A decrease in the amount of fragments found during or
after treatment compared to the amount found before treatment was
started indicates some efficacy of the treatment in ameliorating
the condition of the individual.
[0283] Current methods of assessing the efficacy of various
anti-angiogenic therapies being tested in clinical trials are
invasive. The selective expression of ECSM4 on endothelial cells of
angiogenic blood vessels means that detecting the presence,
absence, increase or decrease in the level of ECSM1 or ECSM4 in a
subject undergoing therapy is a means of determining the efficacy
of the therapy in that subject without the need, or with a reduced
need, for invasive biopsies, scans and the such like.
[0284] Hence, determination of the level of ECSM1 and or ECSM4
fragments in the blood of an individual undergoing an
anti-angiogenic therapy (such as cancer therapy) may act as a
"surrogate marker of angiogenesis".
[0285] By "peptide fragments derived from ECSM1 or ECSM4" we mean
peptides which have at least 5 consecutive amino acids of the ECSM4
or ECSM1 polypeptide. Typically, the fragments have at least 8
consecutive amino acids, preferably at least 10, more preferably at
least 12 or 15 or 20 or 30 or 40 or 50 consecutive amino acids of
the ECSM4 or ECSM1 polypeptide.
[0286] Methods for detecting the presence of fragments of peptides
derived from larger polypeptides are known in the art.
[0287] A further aspect of the invention provides a method, of
modulating angiogenesis in an individual, the method comprising
administering to the individual ESCM4 or a peptide fragment of
ECSM4 or a ligand of ECSM4 or an antibody which selectively binds
to ECSM4 or ECSM1.
[0288] Preferably, the peptide fragment or ligand or antibody is
one which modulates the activity or function, either directly or
indirectly, of the ECSM4 polypeptide of the individual.
[0289] Preferred antibodies are those as described in more detail
above.
[0290] The production of antibodies which modulate the function of
a polypeptide exposed on the cell surface is known in the art and
is discussed in more detail above. Such antibodies may modulate the
function by imitating the function of the natural ligand and
stimulating the polypeptide into activity or function, or may
modulate the polypeptide function by preventing stimulation of the
polypeptide by the ligand by sterically obscuring the ligand
binding site thereby preventing binding of the natural ligand.
[0291] Delivery of a ligand to magic roundabout might be an
angiogenic inhibitor useful in therapy of cancer or other diseases
involving hyper-angiogenesis. Also, introduction of the ECSM4
polypeptide to endothelial cells by gene therapy using the ECSM4
encoding polynucleotide might alter growth and migration.
[0292] A still further aspect of the invention provides a method of
diagnosing a condition which involves aberrant or excessive growth
of vascular endothelium in an individual comprising obtaining a
sample containing nucleic acid from the individual and contacting
said sample with a polynucleotide which selectively hybridises to a
nucleic acid which encodes the ECSM4 polypeptide or the ECSM1
polypeptide or a fragment or natural variant thereof.
[0293] The method may be used for aiding diagnosis.
[0294] A condition which involves aberrant or excessive growth of
vascular endothelium such as cancer, artherosclerosis, restenosis,
diabetic retinopathy, arthritis, psoriasis, endometriosis,
menorrhagia, haemangiomas and venous malformations may be caused by
a mutation in the nucleic acid which encodes the ECSM1 or ECSM4
polypeptides.
[0295] By "selectively hybridising" is meant that the nucleic acid
has sufficient nucleotide sequence similarity with the said human
DNA or cDNA that it can hybridise under moderately or highly
stringent conditions. As is well known in the art, the stringency
of nucleic acid hybridization depends on factors such as length of
nucleic acid over which hybridisation occurs, degree of identity of
the hybridizing sequences and on factors such as temperature, ionic
strength and CG or AT content of the sequence. Thus, any nucleic
acid which is capable of selectively hybridising as said is useful
in the practice of the invention.
[0296] Nucleic acids which can selectively hybridise to the said
human DNA or cDNA include nucleic acids which have >95% sequence
identity, preferably those with >98%, more preferably those with
>99% sequence identity, over at least a portion of the nucleic
acid with the said human DNA or cDNA. As is well known, human genes
usually contain introns such that, for example, a mRNA or cDNA
derived from a gene within the said human DNA would not match
perfectly along its entire length with the said human DNA but would
nevertheless be a nucleic acid capable of selectively hybridising
to the said human DNA. Thus, the invention specifically includes
nucleic acids which selectively hybridise to an ECSM4 or ECSM1 cDNA
but may not hybridise to an ECSM4 or ECSM1 gene, or vice versa. For
example, nucleic acids which span the intron-exon boundaries of the
ECSM4 or ECSM1 gene may not be able to selectively hybridise to the
ECSM4 or ECSM1 cDNA.
[0297] Typical moderately or highly stringent hybridisation
conditions which lead to selective hybridisation are known in the
art, for example those described in Molecular Cloning, a laboratory
manual, 2nd edition, Sambrook et al (eds), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., USA, incorporated
herein by reference.
[0298] An example of a typical hybridisation solution when a
nucleic acid is immobilised on a nylon membrane and the probe
nucleic acid is .gtoreq.500 bases or base pairs is:
6.times.SSC (saline sodium citrate)
0.5% sodium dodecyl sulphate (SDS)
100 .mu.g/ml denatured, fragmented salmon sperm DNA
[0299] The hybridisation is performed at 68.degree. C. The nylon
membrane, with the nucleic acid immobilised, may be washed at
68.degree. C. in 1.times.SSC or, for high stringency,
0.1.times.SSC.
[0300] 20.times.SSC may be prepared in the following way. Dissolve
175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H.sub.2O.
Adjust the pH to 7.0 with a few drops of a 10 N solution of NaOH.
Adjust the volume to 1 litre with H.sub.2O. Dispense into aliquots.
Sterilize by autoclaving.
[0301] An example of a typical hybridisation solution when a
nucleic acid is immobilised on a nylon membrane and the probe is an
oligonucleotide of between 15 and 50 bases is:
3.0 M trimethylammonium chloride (TMACl)
0.01 M sodium phosphate (pH 6.8)
1 mm EDTA (pH 7.6)
0.5% SDS
100 .mu.g/ml denatured, fragmented salmon sperm DNA
0.1% nonfat dried milk
[0302] The optimal temperature for hybridization is usually chosen
to be 5.degree. C. below the T.sub.i for the given chain length.
T.sub.i is the irreversible melting temperature of the hybrid
formed between the probe and its target sequence. Jacobs et al
(1988) Nucl. Acids Res. 16, 4637 discusses the determination of
T.sub.is. The recommended hybridization temperature for 17-mers in
3 M TMACl is 48-50.degree. C.; for 19-mers, it is 55-57.degree. C.;
and for 20-mers, it is 58-66.degree. C.
[0303] By "nucleic acid which selectively hybridises" is also
included nucleic acids which will amplify DNA from the said region
of human DNA by any of the well known amplification systems such as
those described in more detail below, in particular the polymerase
chain reaction (PCR). Suitable conditions for PCR amplification
include amplification in a suitable 1.times. amplification
buffer:
10.times. amplification buffer is 500 mM KCl; 100 mM Tris.Cl (pH
8.3 at room temperature); 15 mM MgCl.sub.2; 0.1% gelatin.
[0304] A suitable denaturing agent or procedure (such as heating to
95.degree. C.) is used in order to separate the strands of
double-stranded DNA.
[0305] Suitably, the annealing part of the amplification is between
37.degree. C. and 60.degree. C., preferably 50.degree. C.
[0306] Although the nucleic acid which is useful in the methods of
the invention may be RNA or DNA, DNA is preferred. Although the
nucleic acid which is useful in the methods of the invention may be
double-stranded or single-stranded, single-stranded nucleic acid is
preferred under some circumstances such as in nucleic acid
amplification reactions.
[0307] The sample may be directly derived from the patient, for
example, by biopsy of a tissue which may be associated with
aberrant vascular development, or it may be derived from the
patient from a site remote from the tissue, for example because
cells from the tissue have migrated from the tissue to other parts
of the body. Alternatively, the sample may be indirectly derived
from the patient in the sense that, for example, the tissue or
cells therefrom may be cultivated in vitro, or cultivated in a
xenograft model; or the nucleic acid sample may be one which has
been replicated (whether in vitro or in vivo) from nucleic acid
from the original source from the patient. Thus, although the
nucleic acid derived from the patient may have been physically
within the patient, it may alternatively have been copied from
nucleic acid which was physically within the patient. When aberrant
vascular development is believed to be associated with a tumour,
tumour tissue may be taken from the primary tumour or from
metastases.
[0308] It will be appreciated that a useful method of the invention
includes the analysis of mutations in, or the detection of the
presence or absence of, the ECSM4 or ECSM1 gene in any suitable
sample. The sample may suitably be a freshly-obtained sample from
the patient, or the sample may be an historic sample, for example a
sample held in a library of samples.
[0309] Conveniently, the nucleic acid capable of selectively
hybridising to the said human DNA and which is used in the methods
of the invention further comprises a detectable label.
[0310] By "detectable label" is included any convenient radioactive
label such as .sup.32P, .sup.33P or .sup.35S which can readily be
incorporated into a nucleic acid molecule using well known methods;
any convenient fluorescent or chemiluminescent label which can
readily be incorporated into a nucleic acid is also included. In
addition the term "detectable label" also includes a moiety which
can be detected by virtue of binding to another moiety (such as
biotin which can be detected by binding to streptavidin); and a
moiety, such as an enzyme, which can be detected by virtue of its
ability to convert a colourless compound into a coloured compound,
or vice versa (for example, alkaline phosphatase can convert
colourless o-nitrophenylphosphate into coloured o-nitrophenol).
Conveniently, the nucleic acid probe may occupy a certain position
in a fixed assay and whether the nucleic acid hybridises to the
said region of human DNA can be determined by reference to the
position of hybridisation in the fixed assay. The detectable label
may also be a fluorophore-quencher pair as described in Tyagi &
Kramer (1996) Nature Biotechnology 14, 303-308.
[0311] Conveniently, in this method of diagnosis of a condition in
which vascular development is aberrant the nucleic acid which is
capable of the said selective hybridisation (whether labelled with
a detectable label or not) is contacted with a nucleic acid derived
from the patient under hybridising conditions. Suitable hybridising
conditions include those described above.
[0312] This method of diagnosing a condition in which vascular
development is aberrant may involve sequencing of DNA at one or
more of the relevant positions within the relevant region,
including direct sequencing; direct sequencing of PCR-amplified
exons; differential hybridisation of an oligonucleotide probe
designed to hybridise at the relevant positions within the relevant
region (conveniently this uses immobilised oligonucleotide probes
in, so-called, "chip" systems which are well known in the art);
denaturing gel electrophoresis following digestion with an
appropriate restriction enzyme, preferably following amplification
of the relevant DNA regions; S1 nuclease sequence analysis;
non-denaturing gel electrophoresis, preferably following
amplification of the relevant DNA regions; conventional RFLP
(restriction fragment length polymorphism) assays; heteroduplex
analysis; selective DNA amplification using oligonucleotides;
fluorescent in-situ hybridisation (FISH) of interphase chromosomes;
ARMS-PCR (Amplification Refractory Mutation System-PCR) for
specific mutations; cleavage at mismatch sites in hybridised
nucleic acids (the cleavage being chemical or enzymic); SSCP single
strand conformational polymorphism or DGGE (discontinuous or
denaturing gradient gel electrophoresis); analysis to detect
mismatch in annealed normal/mutant PCR-amplified DNA; and protein
truncation assay (translation and transcription of exons--if a
mutation introduces a stop codon a truncated protein product will
result). Other methods may be employed such as detecting changes in
the secondary structure of single-stranded DNA resulting from
changes in the primary sequence, for example, using the cleavase I
enzyme.
[0313] This system is commercially available from GibcoBRL, Life
Technologies, 3 Fountain Drive, Inchinnan Business Park, Paisley
PA4 9RF, Scotland.
[0314] It will be appreciated that the methods of the invention may
also be carried out on "DNA chips". Such "chips" are described in
U.S. Pat. No. 5,445,934 (Affymetrix; probe arrays), WO 96/31622
(Oxford; probe array plus ligase or polymerase extension), and WO
95/22058 (Affymax; fluorescently marked targets bind to oligomer
substrate, and location in array detected); all of these are
incorporated herein by reference.
[0315] Detailed methods of mutation detection are described in
"Laboratory Protocols for Mutation Detection" 1996, ed. Landegren,
Oxford University Press on behalf of HUGO (Human Genome
Organisation).
[0316] It is preferred if RFLP is used for the detection of fairly
large (.gtoreq.500 bp) deletions or insertions. Southern blots may
be used for this method of the invention.
[0317] PCR amplification of smaller regions (maximum 300 bp) to
detect small changes greater than 3-4 bp insertions or deletions
may be preferred. Amplified sequence may be analysed on a
sequencing gel, and small changes (minimum size 3-4 bp) can be
visualised. Suitable primers are designed as herein described.
[0318] In addition, using either Southern blot analysis or PCR
restriction enzyme variant sites may be detected. For example, for
analysing variant sites in genomic DNA restriction enzyme
digestion, gel electrophoresis, Southern blotting, and
hybridisation specific probe (for example any suitable fragment
derived from the ECSM4 or ECSM1 cDNA or gene).
[0319] For example, for analysing variant sites using PCR DNA
amplification, restriction enzyme digestion, gel detection by
ethidium bromide, silver staining or incorporation of
radionucleotide or fluorescent primer in the PCR.
[0320] Other suitable methods include the development of allele
specific oligonucleotides (ASOs) for specific mutational events.
Similar methods are used on RNA and cDNA for the suitable
tissue.
[0321] Whilst it is useful to detect mutations in any part of the
ECSM4 or ECSM1 gene, it is preferred if the mutations are detected
in the exons of the gene and it is further preferred if the
mutations are ones which change the coding sense. The detection of
these mutations is a preferred aspect of the invention.
[0322] The methods of the invention also include checking for
loss-of-heterozygosity (LOH; shows one copy lost). LOH may be a
sufficient marker for diagnosis; looking for mutation/loss of the
second allele may not be necessary. LOH of the gene may be detected
using polymorphisms in the coding sequence, and introns, of the
gene.
[0323] Particularly preferred nucleic acids for use in the
aforementioned methods of the invention are those selected from the
group consisting of primers suitable for amplifying nucleic
acid.
[0324] Suitably, the primers are selected from the group consisting
of primers which hybridise to the nucleotide sequences shown in any
of the Figures which show ECSM4 or ECSM1 gene or cDNA sequences. It
is particularly preferred if the primers hybridise to the introns
of the ECSM4 or ECSM1 gene or if the primers are ones which will
prime synthesis of DNA from the ECSM4 or ECSM1 gene or cDNA but not
from other genes or cDNAs.
[0325] Primers which are suitable for use in a polymerase chain
reaction (PCR; Saiki et al (1988) Science 239, 487-491) are
preferred. Suitable PCR primers and methods of detecting products
of PCR reactions are described in detail above.
[0326] Any of the nucleic acid amplification protocols can be used
in the method of the invention including the polymerase chain
reaction, QB replicase and ligase chain reaction. Also, NASBA
(nucleic acid sequence based amplification), also called 3SR, can
be used as described in Compton (1991) Nature 350, 91-92 and AIDS
(1993), Vol 7 (Suppl 2), S108 or SDA (strand displacement
amplification) can be used as described in Walker et al (1992)
Nucl. Acids Res. 20, 1691-1696. The polymerase chain reaction is
particularly preferred because of its simplicity.
[0327] The present invention provides the use of a nucleic acid
which selectively hybridises to the human-derived DNA of genomic
clones as described in Table 8 of Example 1 or to the ECSM4 or
ECSM1 gene, or a mutant allele thereof, or a nucleic acid which
selectively hybridises to ECSM4 or ECSM1 cDNA or a mutant allele
thereof, or their complement in a method of diagnosing a condition
in which vascular development is aberrant; or in the manufacture of
a reagent for carrying out these methods.
[0328] Preferred polynucleotides which selectively hybridise to the
ECSM4 gene or cDNA are as described above in relation to a method
of diagnosis.
[0329] Also, the present invention provides a method of determining
the presence or absence, or mutation in, the said ECSM4 or ECSM1
gene. Preferably, the method uses a suitable sample from a
patient.
[0330] The methods of the invention include the detection of
mutations in the ECSM4 or ECSM1 gene.
[0331] The methods of the invention may make use of a difference in
restriction enzyme cleavage sites caused by mutation. A
non-denaturing gel may be used to detect differing lengths of
fragments resulting from digestion with an appropriate restriction
enzyme.
[0332] An "appropriate restriction enzyme" is one which will
recognise and cut the wild-type sequence and not the mutated
sequence or vice versa. The sequence which is recognised and cut by
the restriction enzyme (or not, as the case may be) can be present
as a consequence of the mutation or it can be introduced into the
normal or mutant allele using mismatched oligonucleotides in the
PCR reaction. It is convenient if the enzyme cuts DNA only
infrequently, in other words if it recognises a sequence which
occurs only rarely.
[0333] In another method, a pair of PCR primers are used which
match (ie hybridise to) either the wild-type genotype or the mutant
genotype but not both. Whether amplified DNA is produced will then
indicate the wild-type or mutant genotype (and hence phenotype).
However, this method relies partly on a negative result (ie the
absence of amplified DNA) which could be due to a technical
failure. It therefore may be less reliable and/or requires
additional control experiments.
[0334] A preferable method employs similar PCR primers but, as well
as hybridising to only one of the wild-type or mutant sequences,
they introduce a restriction site which is not otherwise there in
either the wild-type or mutant sequences.
[0335] The nucleic acids which selectively hybridise to the ECSM4
or ECSM1 gene or cDNA, or which selectively hybridise to the
genomic clones containing ECSM4 or ECSM1 as listed in Table 8 of
Example 1 are useful for a number of purposes. They can be used in
Southern hybridization to genomic DNA and in the RNase protection
method for detecting point mutations already discussed above. The
probes can be used to detect PCR amplification products. They may
also be used to detect mismatches with the ECSM4 or ECSM1 gene or
mRNA in a sample using other techniques. Mismatches can be detected
using either enzymes (eg S1 nuclease or resolvase), chemicals (eg
hydroxylamine or osmium tetroxide and piperidine), or changes in
electrophoretic mobility of mismatched hybrids as compared to
totally matched hybrids. These techniques are known in the art.
Generally, the probes are complementary to the ECSM4 or ECSM1 gene
coding sequences, although probes to certain introns are also
contemplated. A battery of nucleic acid probes may be used to
compose a kit for detecting loss of or mutation in the wild-type
ECSM4 or ECSM1 gene. The kit allows for hybridization to the entire
ECSM4 or ECSM1 gene. The probes may overlap with each other or be
contiguous.
[0336] If a riboprobe is used to detect mismatches with mRNA, it is
complementary to the mRNA of the human ECSM4 or ECSM1 gene. The
riboprobe thus is an anti-sense probe in that it does not code for
the protein encoded by the ECSM4 or ECSM1 gene because it is of the
opposite polarity to the sense strand. The riboprobe generally will
be labelled, for example, radioactively labelled which can be
accomplished by any means known in the art. If the riboprobe is
used to detect mismatches with DNA it can be of either polarity,
sense or anti-sense. Similarly, DNA probes also may be used to
detect mismatches.
[0337] Nucleic acid probes may also be complementary to mutant
alleles of the ECSM4 or ECSM1 gene. These are useful to detect
similar mutations in other patients on the basis of hybridization
rather than mismatches. As mentioned above, the ECSM4 or ECSM1 gene
probes can also be used in Southern hybridizations to genomic DNA
to detect gross chromosomal changes such as deletions and
insertions.
[0338] Particularly useful methods of detecting a mutation in the
ECSM1 or ECSM4 genes include single strand conformation
polymorphism (SSCP), hetero duplex analysis, polymerase chain
reaction, using DNA chips and sequencing.
[0339] Any sample containing nucleic acid derived from the
individual is useful in the methods of the invention. It is
preferred if the nucleic acid in the sample is DNA. Thus, samples
from cells may be obtained as is well known in the art, for example
from blood samples or cheek cells or the like. Where the methods
are being used to determine the presence or absence of a mutation
in an unborn child, it is preferred if the sample is a maternal
sample containing nucleic acid from the unborn child. Suitable
maternal samples include the amniotic fluid of the mother,
chorionic villus samples and blood samples from which foetal cells
can be isolated.
[0340] A further aspect of the invention provides a method of
reducing the expression of the ECSM4 or ECSM1 polynucleotide in an
individual, comprising administering to the individual an agent
which selectively prevents expression of ECSM4 or ECSM1.
[0341] In a preferred embodiment, the agent which selectively
prevents expression of ECSM4 or ECSM1 is an antisense nucleic
acid.
[0342] Preferably, the antisense nucleic acid is not one (or is not
antisense to one) whose sequence consists of the sequence
represented by SEQ ID No 18084 or 5096 of EP 1 074 617, SEQ ID No
210 of WO 00/53756 or WO 99/46281, or SEQ ID Nos 22, 23, 96 or 98
of WO 01/23523 or SEQ ID No 31 of WO 99/11293 or their complement,
or a nucleic acid sequence which encodes a polypeptide whose amino
acid sequence is represented by any one of SEQ ID No 18085 of EP 1
074 617, SEQ ID, No 211 of either WO 00/53756 or WO99/46281, SEQ ID
Nos 24-27, 29, 30, 33, 34, 38 or 39 of WO 01/23523, or SEQ ID No 86
of WO 99/11293.
[0343] A further aspect thereof includes administering an antisense
nucleic acid to a cell in order to prevent expression of ECSM4 or
ECSM1. Typically, the cell is in the body of an individual in need
of prevention of expression of ES CM4 or ECSM1.
[0344] The ECSM4 or ECSM1 polynucleotide which is bound by an
antisense molecule may be DNA or RNA.
[0345] Preferred antisense molecules are as described above.
[0346] Diseases which may be treated by reducing ECSM4 or ECSM1
expression include diseases involving aberrant or excessive
vascularisation as described above.
[0347] Antisense nucleic acids are well known in the art and are
typically single-stranded nucleic acids, which can specifically
bind to a complementary nucleic acid sequence. By binding to the
appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA
duplex is formed. These nucleic acids are often termed "antisense"
because they are complementary to the sense or coding strand of the
gene. Recently, formation of a triple helix has proven possible
where the oligonucleotide is bound to a DNA duplex. It was found
that oligonucleotides could recognise sequences in the major groove
of the DNA double helix. A triple helix was formed thereby. This
suggests that it is possible to synthesise a sequence-specific
molecules which specifically bind double-stranded DNA via
recognition of major groove hydrogen binding sites.
[0348] By binding to the target nucleic acid, the above
oligonucleotides can inhibit the function of the target nucleic
acid. This could, for example, be a result of blocking the
transcription, processing, poly(A)addition, replication,
translation, or promoting inhibitory mechanisms of the cells, such
as promoting RNA degradations.
[0349] Antisense oligonucleotides are prepared in the laboratory
and then introduced into cells, for example by microinjection or
uptake from the cell culture medium into the cells, or they are
expressed in cells after transfection with plasmids or retroviruses
or other vectors carrying an antisense gene. Antisense
oligonucleotides were first discovered to inhibit viral replication
or expression in cell culture for Rous sarcoma virus, vesicular
stomatitis virus, herpes simplex virus type 1, simian virus and
influenza virus. Since then, inhibition of mRNA translation by
antisense oligonucleotides has been studied extensively in
cell-free systems including rabbit reticulocyte lysates and wheat
germ extracts. Inhibition of viral function by antisense
oligonucleotides has been demonstrated in vitro using
oligonucleotides which were complementary to the AIDS HIV
retrovirus RNA (Goodchild, J. 1988 "Inhibition of Human
Immunodeficiency Virus Replication by Antisense
Oligodeoxynucleotides", Proc. Natl. Acad. Sci. (USA) 85(15),
5507-11). The Goodchild study showed that oligonucleotides that
were most effective were complementary to the poly(A) signal; also
effective were those targeted at the 5' end of the RNA,
particularly the cap and 5' untranslated region, next to the primer
binding site and at the primer binding site. The cap, 5'
untranslated region, and poly(A) signal lie within the sequence
repeated at the ends of retrovirus RNA (R region) and the
oligonucleotides complementary to these may bind twice to the
RNA.
[0350] Typically, antisense oligonucleotides are 15 to 35 bases in
length. For example, 20-mer oligonucleotides have been shown to
inhibit the expression of the epidermal growth factor receptor mRNA
(Witters et al, Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer
oligonucleotides have been shown to decrease the expression of
adrenocorticotropic hormone by greater than 90% (Frankel et al, J
Neurosurg 91:261-7 (1999)). However, it is appreciated that it may
be desirable to use oligonucleotides with lengths outside this
range, for example 10, 11, 12, 13, or 14 bases, or 36, 37, 38, 39
or 40 bases.
[0351] Oligonucleotides are subject to being degraded or
inactivated by cellular endogenous nucleases. To counter this
problem, it is possible to use modified oligonucleotides, eg having
altered internucleotide linkages, in which the naturally occurring
phosphodiester linkages have been replaced with another linkage.
For example, Agrawal et al (1988) Proc. Natl. Acad. Sci. USA 85,
7079-7083 showed increased inhibition in tissue culture of HIV-1
using oligonucleotide phosphoramidates and phosphorothioates. Sarin
et al (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451 demonstrated
increased inhibition of HIV-1 using oligonucleotide
methylphosphonates. Agrawal et al (1989) Proc Natl. Acad. Sci. USA
86, 7790-7794 showed inhibition of HIV-1 replication in both
early-infected and chronically infected cell cultures, using
nucleotide sequence-specific oligonucleotide phosphorothioates.
Leither et al (1990) Proc. Natl. Acad. Sci. USA 87, 3430-3434
report inhibition in tissue-culture of influenza virus replication
by oligonucleotide phosphorothioates.
[0352] Oligonucleotides having artificial linkages have been shown
to be resistant to degradation in vivo. For example, Shaw et al
(1991) in Nucleic Acids Res. 19, 747-750, report that otherwise
unmodified oligonucleotides become more resistant to nucleases in
vivo when they are blocked at the 3' end by certain capping
structures and that uncapped oligonucleotide phosphorothioates are
not degraded in vivo.
[0353] A detailed description of the H-phosphonate approach to
synthesizing oligonucleoside phosphorothioates is provided in
Agrawal and Tang (1990) Tetrahedron Letters 31, 7541-7544, the
teachings of which are hereby incorporated herein by reference.
Syntheses of oligonucleoside methylphosphonates,
phosphorodithioates, phosphoramidates, phosphate esters, bridged
phosphoramidates and bridge phosphorothioates are known in the art.
See, for example, Agrawal and Goodchild (1987) Tetrahedron Letters
28, 3539; Nielsen et al (1988) Tetrahedron Letters 29, 2911; Jager
et al (1988) Biochemistry 27, 7237; Uznanski et al (1987)
Tetrahedron Letters 28, 3401; Bannwarth (1988) Helv. Chim. Acta.
71, 1517; Crosstick and Vyle (1989) Tetrahedron Letters 30, 4693;
Agrawal et al (1990) Proc. Natl. Acad. Sci. USA 87, 1401-1405, the
teachings of which are incorporated herein by reference. Other
methods for synthesis or production also are possible. In a
preferred embodiment the oligonucleotide is a deoxyribonucleic acid
(DNA), although ribonucleic acid (RNA) sequences may also be
synthesized and applied.
[0354] The oligonucleotides useful in the invention preferably are
designed to resist degradation by endogenous nucleolytic enzymes.
In vivo degradation of oligonucleotides produces oligonucleotide
breakdown products of reduced length. Such breakdown products are
more likely to engage in non-specific hybridization and are less
likely to be effective, relative to their full-length counterparts.
Thus, it is desirable to use oligonucleotides that are resistant to
degradation in the body and which are able to reach the targeted
cells. The present oligonucleotides can be rendered more resistant
to degradation in vivo by substituting one or more internal
artificial internucleotide linkages for the native phosphodiester
linkages, for example, by replacing phosphate with sulphur in the
linkage. Examples of linkages that may be used include
phosphorothioates, methylphosphonates, sulphone, sulphate, ketyl,
phosphorodithioates, various phosphoramidates, phosphate esters,
bridged phosphorothioates and bridged phosphoramidates. Such
examples are illustrative, rather than limiting, since other
internucleotide linkages are known in the art. See, for example,
Cohen, (1990) Trends in Biotechnology. The synthesis of
oligonucleotides having one or more of these linkages substituted
for the phosphodiester internucleotide linkages is well known in
the art, including synthetic pathways for producing
oligonucleotides having mixed internucleotide linkages.
[0355] Oligonucleotides can be made resistant to extension by
endogenous enzymes by "capping" or incorporating similar groups on
the 5' or 3' terminal nucleotides. A reagent for capping is
commercially available as Amino-Link II.TM. from Applied BioSystems
Inc, Foster City, Calif. Methods for capping are described, for
example, by Shaw et al (1991) Nucleic Acids Res. 19, 747-750 and
Agrawal et al (1991) Proc. Natl. Acad. Sci. USA 88(17), 7595-7599,
the teachings of which are hereby incorporated herein by
reference.
[0356] A further method of making oligonucleotides resistant to
nuclease attack is for them to be "self-stabilized" as described by
Tang et al (1993) Nucl. Acids Res. 21, 2729-2735 incorporated
herein by reference. Self-stabilized oligonucleotides have hairpin
loop structures at their 3' ends, and show increased resistance to
degradation by snake venom phosphodiesterase, DNA polymerase I and
fetal bovine serum. The self-stabilized region of the
oligonucleotide does not interfere in hybridization with
complementary nucleic acids, and pharmacokinetic and stability
studies in mice have shown increased in vivo persistence of
self-stabilized oligonucleotides with respect to their linear
counterparts.
[0357] In accordance with the invention, the antisense compound may
be administered systemically. Alternatively the inherent binding
specificity of antisense oligonucleotides characteristic of base
pairing is enhanced by limiting the availability of the antisense
compound to its intended locus in vivo, permitting lower dosages to
be used and minimising systemic effects. Thus, oligonucleotides may
be applied locally to achieve the desired effect. The concentration
of the oligonucleotides at the desired locus is much higher than if
the oligonucleotides were administered systemically, and the
therapeutic effect can be achieved using a significantly lower
total amount. The local high concentration of oligonucleotides
enhances penetration of the targeted cells and effectively blocks
translation of the target nucleic acid sequences.
[0358] The oligonucleotides can be delivered to the locus by any
means appropriate for localised administration of a drug. For
example, a solution of the oligonucleotides can be injected
directly to the site or can be delivered by infusion using an
infusion pump. The oligonucleotides also can be incorporated into
an implantable device which when placed at the desired site,
permits the oligonucleotides to be released into the surrounding
locus.
[0359] The oligonucleotides may be administered via a hydrogel
material. The hydrogel is non-inflammatory and biodegradable. Many
such materials now are known, including those made from natural and
synthetic polymers. In a preferred embodiment, the method exploits
a hydrogel which is liquid below body temperature but gels to form
a shape-retaining semisolid hydrogel at or near body temperature.
Preferred hydrogel are polymers of ethylene oxide-propylene oxide
repeating units. The properties of the polymer are dependent on the
molecular weight of the polymer and the relative percentage of
polyethylene oxide and polypropylene oxide in the polymer.
Preferred hydrogels contain from about 10% to about 80% by weight
ethylene oxide and from about 20% to about 90% by weight propylene
oxide. A particularly preferred hydrogel contains about 70%
polyethylene oxide and 30% polypropylene oxide. Hydrogels which can
be used are available, for example, from BASF Corp., Parsippany,
N.J., under the tradename Pluronic.sup.R.
[0360] In this embodiment, the hydrogel is cooled to a liquid state
and the oligonucleotides are admixed into the liquid to a
concentration of about 1 mg oligonucleotide per gram of hydrogel.
The resulting mixture then is applied onto the surface to be
treated, for example by spraying or painting during surgery or
using a catheter or endoscopic procedures. As the polymer warms, it
solidifies to form a gel, and the oligonucleotides diffuse out of
the gel into the surrounding cells over a period of time defined by
the exact composition of the gel.
[0361] It will be appreciated that the oligonucleotides or other
agents may be administered after surgical removal of a tumour, and
may be administered to the area from which the tumour has been
removed, and surrounding tissue, for example using cytoscopy to
guide application of the oligonucleotides or other agents.
[0362] The oligonucleotides can be administered by means of other
implants that are commercially available or described in the
scientific literature, including liposomes, microcapsules and
implantable devices. For example, implants made of biodegradable
materials such as polyanhydrides, polyorthoesters, polylactic acid
and polyglycolic acid and copolymers thereof, collagen, and protein
polymers, or non-biodegradable materials such as ethylenevinyl
acetate (EVAc), polyvinyl acetate, ethylene vinyl alcohol, and
derivatives thereof can be used to locally deliver the
oligonucleotides. The oligonucleotides can be incorporated into the
material as it is polymerised or solidified, using melt or solvent
evaporation techniques, or mechanically mixed with the material. In
one embodiment, the oligonucleotides are mixed into or applied onto
coatings for implantable devices such as dextran coated silica
beads, stents, or catheters.
[0363] The dose of oligonucleotides is dependent on the size of the
oligonucleotides and the purpose for which is it administered. In
general, the range is calculated based on the surface area of
tissue to be treated. The effective dose of oligonucleotide is
somewhat dependent on the length and chemical composition of the
oligonucleotide but is generally in the range of about 30 to 3000
.mu.g per square centimetre of tissue surface area.
[0364] The oligonucleotides may be administered to the patient
systemically for both therapeutic and prophylactic purposes. The
oligonucleotides may be administered by any effective method, for
example, parenterally (eg intravenously, subcutaneously,
intramuscularly) or by oral, nasal or other means which permit the
oligonucleotides to access and circulate in the patient's
bloodstream. Oligonucleotides administered systemically preferably
are given in addition to locally administered oligonucleotides, but
also have utility in the absence of local administration. A dosage
in the range of from about 0.1 to about 10 grams per administration
to an adult human generally will be effective for this purpose.
[0365] It will be appreciated that antisense agents also include
larger molecules which bind to said ECSM4 or ECSM1 mRNA or genes
and substantially prevent expression of said ECSM4 or ECSM1 mRNA or
genes and substantially prevent expression of said ECSM4 or ECSM1
protein. Thus, expression of an antisense molecule which is
substantially complementary to said ECSM4 or ECSM1 mRNA is
envisaged as part of the invention.
[0366] The said larger molecules may be expressed from any suitable
genetic construct as is described below and delivered to the
patient. Typically, the genetic construct which expresses the
antisense molecule comprises at least a portion of the said ECSM4
or ECSM1 cDNA or gene operatively linked to a promoter which can
express the antisense molecule in a cell. Promoters that may be
active in endothelial cells are described below.
[0367] Although the genetic construct can be DNA or RNA it is
preferred if it is DNA.
[0368] Preferably, the genetic construct is adapted for delivery to
a human cell.
[0369] Means and methods of introducing a genetic construct into a
cell in an animal body are known in the art. For example, the
constructs of the invention may be introduced into proliferating
endothelial cells by any convenient method, for example methods
involving retroviruses, so that the construct is inserted into the
genome of the endothelial cell. For example, in Kuriyama et al
(1991) Cell Struc. and Func. 16, 503-510 purified retroviruses are
administered. Retroviruses provide a potential means of selectively
infecting proliferating endothelial cells because they can only
integrate into the genome of dividing cells; most endothelial cells
are in a quiescent, non-receptive stage of cell growth or, at
least, are dividing much less rapidly than angiogenic cells.
Retroviral DNA constructs which encode said antisense agents may be
made using methods well known in the art. To produce active
retrovirus from such a construct it is usual to use an ecotropic
psi2 packaging cell line grown in Dulbecco's modified Eagle's
medium (DMEM) containing 10% foetal calf serum (FCS). Transfection
of the cell line is conveniently by calcium phosphate
co-precipitation, and stable transformants are selected by addition
of G418 to a final concentration of 1 mg/ml (assuming the
retroviral construct contains a neo.sup.R gene). Independent
colonies are isolated and expanded and the culture supernatant
removed, filtered through a 0.45 .mu.m pore-size filter and stored
at -70.degree.. For the introduction of the retrovirus into the
tumour cells, it is convenient to inject directly retroviral
supernatant to which 10 .mu.g/ml Polybrene has been added. For
tumours exceeding 10 mm in diameter it is appropriate to inject
between 0.1 ml and 1 ml of retroviral supernatant; preferably 0.5
ml.
[0370] Alternatively, as described in Culver et al (1992) Science
256, 1550-1552, cells which produce retroviruses are injected into
specific tissue. The retrovirus-producing cells so introduced are
engineered to actively produce retroviral vector particles so that
continuous productions of the vector occurred within the tumour
mass in situ. Thus, proliferating endothelial cells can be
successfully transduced in vivo if mixed with retroviral
vector-producing cells.
[0371] Targeted retroviruses are also available for use in the
invention; for example, sequences conferring specific binding
affinities may be engineered into pre-existing viral env genes (see
Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this
and other targeted vectors for gene therapy).
[0372] Other methods involve simple delivery of the construct into
the cell for expression therein either for a limited time or,
following integration into the genome, for a longer time. An
example of the latter approach includes (preferably
endothelial-cell-targeted) liposomes (Nassander et al (1992) Cancer
Res. 52, 646-653).
[0373] Immunoliposomes (antibody-directed liposomes) are especially
useful in targeting to endothelial cell types which express a cell
surface protein for which antibodies are available.
[0374] Other methods of delivery include adenovinises carrying
external DNA via an antibody-polylysine bridge (see Curiel Prog.
Med. Virol. 40, 1-18) and transferrin-polycation conjugates as
carriers (Wagner et al (1990) Proc. Natl. Acad. Sci. USA 87,
3410-3414). In the first of these methods a polycation-antibody
complex is formed with the DNA construct or other genetic construct
of the invention, wherein the antibody is specific for either
wild-type adenovirus or a variant adenovirus in which a new epitope
has been introduced which binds the antibody. The polycation moiety
binds the DNA via electrostatic interactions with the phosphate
backbone. The adenovirus, because it contains unaltered fibre and
penton proteins, is internalised into the cell and carries into the
cell with it the DNA construct of the invention. It is preferred if
the polycation is polylysine.
[0375] The DNA may also be delivered by adenovirus wherein it is
present within the adenovirus particle, for example, as described
below.
[0376] In the second of these methods, a high-efficiency nucleic
acid delivery system that uses receptor-mediated endocytosis to
carry DNA macromolecules into cells is employed. This is
accomplished by conjugating the iron-transport protein transferrin
to polycations that bind nucleic acids. Human transferrin, or the
chicken homologue conalbumin, or combinations thereof is covalently
linked to the small DNA-binding protein protamine or to polylysines
of various sizes through a disulfide linkage. These modified
transferrin molecules maintain their ability to bind their cognate
receptor and to mediate efficient iron transport into the cell. The
transferrin-polycation molecules form electrophoretically stable
complexes with DNA constructs or other genetic constructs of the
invention independent of nucleic acid size (from short
oligonucleotides to DNA of 21 kilobase pairs). When complexes of
transferrin-polycation and the DNA constructs or other genetic
constructs of the invention are supplied to the endothelial cells,
a high level of expression from the construct in the cells is
expected.
[0377] High-efficiency receptor-mediated delivery of the DNA
constructs or other genetic constructs of the invention using the
endosome-disruption activity of defective or chemically inactivated
adenovirus particles produced by the methods of Cotten et al (1992)
Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used. This
approach appears to rely on the fact that adenoviruses are adapted
to allow release of their DNA from an endosome without passage
through the lysosome, and in the presence of, for example
transferrin linked to the DNA construct or other genetic construct
of the invention, the construct is taken up by the cell by the same
route as the adenovirus particle.
[0378] This approach has the advantages that there is no need to
use complex retroviral constructs; there is no permanent
modification of the genome as occurs with retroviral infection; and
the targeted expression system is coupled with a targeted delivery
system, thus reducing toxicity to other cell types.
[0379] It may be desirable to locally perfuse a tumour with the
suitable delivery vehicle comprising the genetic construct for a
period of time; additionally or alternatively the delivery vehicle
or genetic construct can be injected directly into accessible
tumours.
[0380] It will be appreciated that "naked DNA" and DNA complexed
with cationic and neutral lipids may also be useful in introducing
the DNA of the invention into cells of the patient to be treated.
Non-viral approaches to gene therapy are described in Ledley (1995)
Human Gene Therapy 6, 1129-1144.
[0381] Alternative targeted delivery systems are also known such as
the modified adenovirus system described in WO 94/10323 wherein,
typically, the DNA is carried within the adenovirus, or
adenovirus-like, particle. Michael et al (1995) Gene Therapy 2,
660-668 describes modification of adenovirus to add a
cell-selective moiety into a fibre protein. Mutant adenoviruses
which replicate selectively in p53-deficient human tumour cells,
such as those described in Bischoff et al (1996) Science 274,
373-376 are also useful for delivering the genetic construct of the
invention to a cell. Thus, it will be appreciated that a further
aspect of the invention provides a virus or virus-like particle
comprising a genetic construct of the invention. Other suitable
viruses or virus-like particles include HSV, AAV, vaccinia and
parvovirus.
[0382] In a further embodiment the agent which selectively prevents
the function of ECSM4 or ECSM1 is a ribozyme capable of cleaving
targeted ECSM4 or ECSM1 RNA or DNA. A gene expressing said ribozyme
may be administered in substantially the same and using
substantially the same vehicles as for the antisense molecules.
[0383] Ribozymes which may be encoded in the genomes of the viruses
or virus-like particles herein disclosed are described in Cech and
Herschlag "Site-specific cleavage of single stranded DNA" U.S. Pat.
No. 5,180,818; Altman et al "Cleavage of targeted RNA by RNAse P"
U.S. Pat. No. 5,168,053, Cantin et al "Ribozyme cleavage of HIV-1
RNA" U.S. Pat. No. 5,149,796; Cech et al "RNA ribozyme restriction
endoribonucleases and methods", U.S. Pat. No. 5,116,742; Been et al
"RNA ribozyme polymerases, dephosphorylases, restriction
endonucleases and methods", U.S. Pat. No. 5,093,246; and Been et al
"RNA ribozyme polymerases, dephosphorylases, restriction
endoribonucleases and methods; cleaves single-stranded RNA at
specific site by transesterification", U.S. Pat. No. 4,987,071, all
incorporated herein by reference.
[0384] It will be appreciated that it may be desirable that the
antisense molecule or ribozyme is expressed from a cell-specific
promoter element.
[0385] The genetic constructs of the invention can be prepared
using methods well known in the art.
[0386] A further aspect of the invention is a method of screening
for a molecule that binds to ECSM4 or a suitable variant, fragment
or fusion thereof, or a fusion of a said fragment or fusion
thereof, the method comprising 1) contacting a) the ECSM4
polypeptide with b) a test molecule 2) detecting the presence of a
complex containing the ECSM4 polypeptide and a test molecule, and
optionally 3) identifying any test molecule bound to the ECSM4
polypeptide.
[0387] Preferably the ECSM4 polypeptide is one as described above
in respect of the eleventh aspect of the invention.
[0388] In a preferred embodiment, the test molecule is a
polypeptide.
[0389] In a further preferred embodiment, the method is used to
identify natural ligands of ECSM4. Thus, in this embodiment the
test molecule includes the natural ligand of ECSM4. A particularly
useful technique for the identification of natural ligands of
polypeptide molecules is the yeast two-hybrid technique. This
technique is well known in the art and relies on binding between a
molecule and its cognate ligand to bring together two parts of a
transcription complex (which are fused one to the molecule in
question and other to the test ligand) which, when together,
promote transcription of a reporter gene.
[0390] Hence, a preferred embodiment of this aspect of the
invention comprises use of the screening method, preferably the
yeast two-hybrid system, to identify natural ligands of the ECSM4
polypeptide.
[0391] A molecule which is identifiable as binding the ECSM4
polypeptide is a further aspect of the invention.
[0392] It will be appreciated that a molecule which binds to ESCM4
may modulate the activation of ECSM4.
[0393] Suitable peptide ligands that will bind to ECSM4 may be
identified using methods known in the art.
[0394] One method, disclosed by Scott and Smith (1990) Science 249,
386-390 and Cwirla et al (1990) Proc. Natl. Acad. Sci. USA 87,
6378-6382, involves the screening of a vast library of filamentous
bacteriophages, such as M13 or fd, each member of the library
having a different peptide fused to a protein on the surface of the
bacteriophage. Those members of the library that bind to ECSM4 are
selected using an iterative binding protocol, and once the phages
that bind most tightly have been purified, the sequence of the
peptide ligands may be determined simply by sequencing the DNA
encoding the surface protein fusion. Another method that can be
used is the NovaTope.TM. system commercially available from
Novagen, Inc., 597 Science Drive, Madison, Wis. 53711. The method
is based on the creation of a library of bacterial clones, each of
which stably expresses a small peptide derived from a candidate
protein in which the ligand is believed to reside. The library is
screened by standard lift methods using the antibody or other
binding agent as a probe. Positive clones can be analysed directly
by DNA sequencing to determine the precise amino acid sequence of
the ligand.
[0395] Further methods using libraries of beads conjugated to
individual species of peptides as disclosed by Lam et al (1991)
Nature 354, 82-84 or synthetic peptide combinatorial libraries as
disclosed by Houghten et al (1991) Nature 354, 84-86 or matrices of
individual synthetic peptide sequences on a solid support as
disclosed by Pirrung et al in U.S. Pat. No. 5,143,854 may also be
used to identify peptide ligands.
[0396] It will be appreciated that screening assays which are
capable of high throughput operation will be particularly
preferred. Examples may include cell based assays and
protein-protein binding assays. An SPA-based (Scintillation
Proximity Assay; Amersham International) system may be used. For
example, an assay for identifying a compound capable of modulating
the activity of a protein kinase may be performed as follows. Beads
comprising scintillant and a polypeptide that may be phosphorylated
may be prepared. The beads may be mixed with a sample comprising
the protein kinase and .sup.32P-ATP or .sup.33P-ATP and with the
test compound. Conveniently this is done in a 96-well format. The
plate is then counted using a suitable scintillation counter, using
known parameters for .sup.32P or .sup.33P SPA assays. Only .sup.32P
or .sup.33P that is in proximity to the scintillant, i.e. only that
bound to the polypeptide, is detected. Variants of such an assay,
for example in which the polypeptide is immobilised on the
scintillant beads via binding to an antibody, may also be used.
[0397] Other methods of detecting polypeptide/polypeptide
interactions include ultrafiltration with ion spray mass
spectroscopy/HPLC methods or other physical and analytical methods.
Fluorescence Energy Resonance Transfer (FRET) methods, for example,
well known to those skilled in the art, may be used, in which
binding of two fluorescent labelled entities may be measured by
measuring the interaction of the fluorescent labels when in close
proximity to each other.
[0398] Alternative methods of detecting binding of a polypeptide to
macromolecules, for example DNA, RNA, proteins and phospholipids,
include a surface plasmon resonance assay, for example as described
in Plant et al (1995) Analyt Biochem 226(2), 342-348. Methods may
make use of a polypeptide that is labelled, for example with a
radioactive or fluorescent label.
[0399] A further method of identifying a compound that is capable
of binding to the ECSM4 polypeptide is one where the polypeptide is
exposed to the compound and any binding of the compound to the said
polypeptide is detected and/or measured. The binding constant for
the binding of the compound to the polypeptide may be determined.
Suitable methods for detecting and/or measuring (quantifying) the
binding of a compound to a polypeptide are well known to those
skilled in the art and may be performed, for example, using a
method capable of high throughput operation, for example a
chip-based method. New technology, called VLSIPS.TM., has enabled
the production of extremely small chips that contain hundreds of
thousands or more of different molecular probes. These biological
chips or arrays have probes arranged in arrays, each probe assigned
a specific location. Biological chips have been produced in which
each location has a scale of, for example, ten microns. The chips
can be used to determine whether target molecules interact with any
of the probes on the chip. After exposing the array to target
molecules under selected test conditions, scanning devices can
examine each location in the array and determine whether a target
molecule has interacted with the probe at that location.
[0400] Biological chips or arrays are useful in a variety of
screening techniques for obtaining information about either the
probes or the target molecules. For example, a library of peptides
can be used as probes to screen for drugs. The peptides can be
exposed to a receptor, and those probes that bind to the receptor
can be identified. See U.S. Pat. No. 5,874,219 issued 23 Feb. 1999
to Rava et al.
[0401] Another method of targeting proteins that modulate the
activity of ECSM4 is the yeast two-hybrid system, where the
polypeptides of the invention can be used to "capture" ECSM4
protein binding proteins. The yeast two-hybrid system is described
in Fields & Song, Nature 340:245-246 (1989).
[0402] It will be understood that it will be desirable to identify
compounds that may modulate the activity of the polypeptide ini
vivo. Thus it will be understood that reagents and conditions used
in the method may be chosen such that the interactions between the
said and the interacting polypeptide are substantially the same as
between a said naturally occurring polypeptide and a naturally
occurring interacting polypeptide in vivo.
[0403] It will be appreciated that in the method described herein,
the ligand may be a drug-like compound or lead compound for the
development of a drug-like compound.
[0404] The term "drug-like compound" is well known to those skilled
in the art, and may include the meaning of a compound that has
characteristics that may make it suitable for use in medicine, for
example as the active ingredient in a medicament. Thus, for
example, a drug-like compound may be a molecule that may be
synthesised by the techniques of organic chemistry, less preferably
by techniques of molecular biology or biochemistry, and is
preferably a small molecule, which may be of less than 5000 daltons
and which may be water-soluble. A drug-like compound may
additionally exhibit features of selective interaction with a
particular protein or proteins and be bioavailable and/or able to
penetrate target cellular membranes, but it will be appreciated
that these features are not essential.
[0405] The term "lead compound" is similarly well known to those
skilled in the art, and may include the meaning that the compound,
whilst not itself suitable for use as a drug (for example because
it is only weakly potent against its intended target, non-selective
in its action, unstable, poorly soluble, difficult to synthesise or
has poor bioavailability) may provide a starting-point for the
design of other compounds that may have more desirable
characteristics.
[0406] Alternatively, the methods may be used as "library
screening" methods, a term well known to those skilled in the art.
Thus, for example, the method of the invention may be used to
detect (and optionally identify) a polynucleotide capable of
expressing a polypeptide activator of ECSM4. Aliquots of an
expression library in a suitable vector may be tested for the
ability to give the required result.
[0407] Hence, an embodiment of this aspect of the invention
provides a method of identifying a drug-like compound or lead
compound for the development of a drug-like compound that modulates
the activity of the polypeptide ECSM4, the method comprising
contacting a compound with the polypeptide or a suitable variant,
fragment, derivative or fusion thereof or a fusion of a variant,
fragment or derivative thereof and determining whether, for
example, the enzymic activity of the said polypeptide is changed
compared to the activity of the said polypeptide or said variant,
fragment, derivative or fusion thereof or a fusion of a variant,
fragment or derivative thereof in the absence of said compound.
[0408] Preferably, the ECSM4 polypeptide is as described above in
respect of the eleventh aspect of the invention.
[0409] It will be understood that it will be desirable to identify
compounds that may modulate the activity of the polypeptide in
vivo. Thus it will be understood that reagents and conditions used
in the method may be chosen such that the interactions between the
said polypeptide and its substrate are substantially the same as in
vivo.
[0410] In one embodiment, the compound decreases the activity of
said polypeptide. For example, the compound may bind substantially
reversibly or substantially irreversibly to the active site of said
polypeptide. In a further example, the compound may bind to a
portion of said polypeptide that is not the active site so as to
interfere with the binding of the said polypeptide to its ligand.
In a still further example, the compound may bind to a portion of
said polypeptide so as to decrease said polypeptide's activity by
an allosteric effect. This allosteric effect may be an allosteric
effect that is involved in the natural regulation of the said
polypeptide's activity, for example in the activation of the said
polypeptide by an "upstream activator".
[0411] A still further aspect of the invention provides a
polynucleotide comprising a promoter and/or regulatory portion of
any one of the ECSM1 or ECSM4 genes.
[0412] By "ECSM1 or ECSM4 genes" we mean the natural genomic
sequence which when transcribed is capable of encoding a
polypeptide comprising the ECSM1 or ECSM4 polypeptide sequence as
defined herein. The natural genomic sequence of the ECSM1 or ECSM4
genes may contain introns.
[0413] The polynucleotide of this aspect of the invention is
preferably one which has transcriptional promoter activity. A
promoter is an expression control element formed by a DNA sequence
that permits binding of RNA polymerase and transcription to occur.
Preferably the transcriptional promoter activity is present in
mammalian cells and more preferably the polynucleotide has
transcriptional promoter activity in endothelial cells. In a
preferred embodiment, the transcriptional promoter activity is
present in endothelial cells and not in other cell types.
[0414] Preferably, the promoter and/or regulatory portion is one
which can direct endothelial cell selective expression.
[0415] Preferably, the promoter or regulatory region of the ECSM4
gene is one which is capable of promoting transcription of an
operatively-linked coding sequence in response to hypoxic
conditions. More preferably, the level of transcription of the
coding sequence is up-regulated in hypoxic conditions compared to
the level of transcription in the absence of hypoxia. By "hypoxic
conditions" we include the physiological conditions of cancer where
the inappropriate cell proliferation deprives surrounding tissue of
oxygen, cardiac disease where for example a vessel occlusion may
restrict the delivery of oxygen to certain tissues, and tissue
necrosis where destruction of vascular tissue cells results in a
reduced supply of oxygen to surrounding tissue and the consequent
death of that surrounding tissue. Hypoxia is described in more
detail in Hockel and Vaupel (2001) J. Nat. Can. Inst. 93:
266-276.
[0416] Hence, in a preferred embodiment, the ECSM4 promoter or
regulatory region is comprised in a vector suitable for use in gene
therapy for driving expression of a therapeutic gene to treat a
hypoxic condition. Preferably, the hypoxic condition is cancer or
cardiac disease. A "therapeutic gene" may be any gene which
provides a desired therapeutic effect.
[0417] It will be appreciated that use of the said ECSM4 promoter
to treat a hypoxic condition, for example by gene therapy, is
included within the scope of the present invention.
[0418] Methods for the determination of the sequence of the
promoter region of a gene are well known in the art. The presence
of a promoter region may be determined by identification of known
motifs, and confirmed by mutational analysis of the identified
sequence. Preferably, the promoter sequence is located in the
region 5 kb upstream of the genomic coding region of ECSM 1 or
ECSM4. More preferably, it is located in the region 3 kb or 2 kb or
1 kb or 500 bp upstream, and still more preferably it is located
within 210 bp of the transcription start site.
[0419] Regulatory regions, or transcriptional elements such as
enhancers are less predictable than promoters in their location
relative to a gene. However, many motifs indicative of regulatory
regions are well characterised and such regions affecting the level
of transcription of the relevant gene can usually be identified on
the basis of these motifs. The function of such a region can be
demonstrated by well-known methods such as mutational analysis and
in vitro DNA-binding assays including DNA footprinting and gel
mobility shift assays.
[0420] Regulatory regions influencing the transcription of the
ECSM1 or ECSM4 genes are likely to be located within the region 20
kb or 10 kb or 7 kb 5 kb or 3 kb, or more preferably 1 kb 5'
upstream of the relevant genomic coding region or can be located
within introns of the gene.
[0421] Sequence tagged sites and mapping intervals will be helpful
in localising promoter regions, regulatory regions and physical
clones.
[0422] In a further preferred embodiment, the polynucleotide
comprising the promoter and/or regulatory portion is operatively
linked to a polynucleotide encoding a polypeptide. Methods for
linking promoter polynucleotides to polypeptide coding sequences
are well known in the art.
[0423] Preferably the polypeptide is a therapeutic polypeptide. A
therapeutic polypeptide may be any polypeptide which it is
medically useful to express selectively in endothelial cells.
Examples of such therapeutic polypeptides include
anti-proliferative, immunomodulatory or blood clotting-influencing
factors, or anti-proliferative or anti-inflammatory cytokines. They
may also comprise anti-cancer polypeptides.
[0424] In one embodiment of this aspect of the invention, the
polynucleotide is one suitable for use in medicine. Thus, the
invention includes the polynucleotide packaged and presented for
use in medicine. It will be appreciated that such polynucleotides
will be especially useful in gene therapy, especially where it is
desirable to express a therapeutic polypeptide selectively an
endothelial cell. It is preferred if the polynucleotide is one
suitable for use in gene therapy.
[0425] Gene therapy may be carried out according to generally
accepted methods, for example, as described by Friedman, 1991. A
virus or plasmid vector (see further details below), containing a
copy of the gene to be expressed linked to expression control
elements such as promoters and other regulatory elements
influencing transcription of ECSM1 or ECSM4 as described above and
capable of replicating inside endothelial cells, is prepared.
Suitable vectors are known, such as disclosed in U.S. Pat. No.
5,252,479 and WO 93/07282. The vector is then injected into the
patient, either locally or systemically. If the transfected gene is
not permanently incorporated into the genome of each of the
targeted endothelial cells, the treatment may have to be repeated
periodically.
[0426] Gene transfer systems known in the art may be useful in the
practice of the gene therapy methods of the present invention.
These include viral and nonviral transfer methods. A number of
viruses have been used as gene transfer vectors, including
papovaviruses, eg SV40 (Madzak et al, 1992), adenovirus (Berkner,
1992; Berkner et al, 1988; Gorziglia and Kapikian, 1992; Quantin et
al, 1992; Rosenfeld et al, 1992; Wilkinson et al, 1992;
Stratford-Perricaudet et al, 1990), vaccinia virus (Moss, 1992),
adeno-associated virus (Muzyczka, 1992; Ohi et al, 1990),
herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et
al, 1992; Fink et al, 1992; Breakfield and Geller, 1987; Freese et
al, 1990), and retroviruses of avian (Brandyopadhyay and Temin,
1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller et
al, 1985; Sorge et al, 1984; Mann and Baltimore, 1985; Miller et
al, 1988), and human origin (Shimada et al, 1991; Helseth et al,
1990; Page et al, 1990; Buchschacher and Panganiban, 1992). To date
most human gene therapy protocols have been based on disabled
murine retroviruses.
[0427] Nonviral gene transfer methods known in the art include
chemical techniques such as calcium phosphate coprecipitation
(Graham and van der Eb, 1973; Pellicer et al, 1980); mechanical
techniques, for example microinjection (Anderson et al, 1980;
Gordon et al, 1980; Brinster et al, 1981; Constantini and Lacy,
1981); membrane fusion-mediated transfer via liposomes (Felgner et
al, 1987; Wang and Huang, 1989; Kaneda et al, 1989; Stewart et al,
1992; Nabel et al, 1990; tim et al, 1992); and direct DNA uptake
and receptor-mediated DNA transfer (Wolff et al, 1990; Wu et al,
1991; Zenke et al, 1990; Wu et al, 1989b; Wolff et al, 1991; Wagner
et al, 1990; Wagner et al, 1991; Cotten et al, 1990; Curiel et al,
1991a; Curiel et al, 1991b).
[0428] Other suitable systems include the retroviral-adenoviral
hybrid system described by Feng et al (1997) Nature Biotechnology
15, 866-870, or viral systems with targeting ligands such as
suitable single chain Fv fragments.
[0429] In an approach which combines biological and physical gene
transfer methods, plasmid DNA of any size is combined with a
polylysine-conjugated antibody specific to the adenovirus hexon
protein, and the resulting complex is bound to an adenovirus
vector. The trimolecular complex is then used to infect cells. The
adenovirus vector permits efficient binding, internalization, and
degradation of the endosome before the coupled DNA is damaged.
[0430] Liposome/DNA complexes have been shown to be capable of
mediating direct in vivo gene transfer. While in standard liposome
preparations the gene transfer process is nonspecific, localized in
vivo uptake and expression have been reported in tumour deposits,
for example, following direct in situ administration (Nabel,
1992).
[0431] Gene transfer techniques which target DNA directly to
tissues, eg endothelial cells, is preferred. Receptor-mediated gene
transfer, for example, is accomplished by the conjugation of DNA
(usually in the form of covalently closed supercoiled plasmid) to a
protein ligand via polylysine. Ligands are chosen on the basis of
the presence of the corresponding ligand receptors on the cell
surface of the target cell/tissue type. In the case of endothelial
cells, a suitable receptor is ECSM4. These ligand-DNA conjugates
can be injected directly into the blood if desired and are directed
to the target tissue where receptor binding and internalization of
the DNA-protein complex occurs. To overcome the problem of
intracellular destruction of DNA, coinfection with adenovirus can
be included to disrupt endosome function.
[0432] In the case where replacement gene therapy using a
functionally wild-type gene is used, it may be useful to monitor
the treatment by detecting the presence of replacement gene mRNA or
encoded replacement polypeptide, or functional gene product, at
various sites in the body, including the endothelial cells, blood
serum, and bodily secretions/excretions, for example urine.
[0433] A further aspect of the present invention provides a method
of treating an individual with cancer, cardiac disease, a hypoxic
condition, endometriosis or artherosclerosis comprising
administering to the individual a polynucleotide according to the
invention, which polynucleotide comprises a promoter or regulatory
region of the invention operatively linked to a polynucleotide
encoding a therapeutic polypeptide.
[0434] A still further aspect of the invention provides a method of
modulating angiogenesis in an individual comprising administering
to the individual a polynucleotide according to the invention,
which polynucleotide comprises a promoter or regulatory region of
the invention operatively linked to a polynucleotide encoding a
therapeutic polypeptide or a polynucleotide which is capable of
expressing ECSM4 or a fragment or variant thereof or which
comprises an ECSM4 antisense nucleic acid.
[0435] The therapeutic polypeptide may be any therapeutic
polypeptide which is useful in treating the individual. Preferably,
the therapeutic polypeptide is any one or more of immunomodulatory,
anti-cancer, a blood-clotting-influencing factor or an
anti-proliferative or anti-inflammatory cytokine.
[0436] Antisense nucleic acid is discussed in more detail above.
Briefly, the function of an antisense nucleic acid is to inhibit
the translation of a specific mRNA to which the antisense nucleic
acid is complementary and able to hybridise to within a cell, at
least in part. The design of optimal antisense nucleic acid
molecules is well known in the art of molecular biology.
[0437] The present invention also provides a use of a
polynucleotide according to the invention, which polynucleotide
comprises a promoter or regulatory region of the invention
operatively linked to a polynucleotide encoding a therapeutic
polypeptide in the manufacture of a medicament for treating cancer,
cardiac disease, a hypoxic condition, endometriosis or
artherosclerosis.
[0438] The invention will now be described in more detail by
reference to the following Examples and Figures herein
[0439] FIG. 1.
[0440] Experimental verification by reverse transcription PCR.
Candidate endothelial specific genes predicted by the combination
of the UniGene/EST screen and xProfiler SAGE differential analysis
(Table 8) were checked for expression in three endothelial and nine
non-endothelial cell cultures. Endothelial cultures were as
follows: HMVEC (human microvascular endothelial cells), HUVEC
(human umbilical vein endothelial cells) confluent culture and
HUVEC proliferating culture. Non-endothelial cultures were as
follows: normal endometrial stromal (NES) cells grown in normoxia
and NES grown in hypoxia, MDA 453 and MDA 468 breast carcinoma cell
lines, HeLa, FEK4 fibroblasts cultured in normoxia and FEK4
fibroblasts cultured in hypoxia, and SW480, HCT116--two colorectal
epithelium cell lines. ECSM1 showed complete endothelial
specificity, while magic roundabout/ECSM4 was very strongly
preferentially expressed in the endothelium. Interestingly, both
these novel genes appear more endothelial specific than the
benchmark endothelial specific gene: von Willebrand factor.
[0441] FIG. 2.
[0442] Phrap generated contig sequence for ECSM1 and amino acid
sequence of the translation product. The ESTs used to generate this
contig are shown in Table 10.
[0443] FIG. 3.
[0444] ECSM4 in vitro transcription/translation. The cDNA coding
for full length ECSM4 was cloned into pBluescript plasmid vector.
Circular and HindIII digested plasmid were subjected to in vitro
transcription/translation using TNT.RTM. T7 Quick Coupled
Transcription/Translation System (Promega Corporation)
incorporating .sup.35S Methionine as per manufacturer's
instructions. The reaction products were resolved by SDS PAGE and
visualised by autoradiography. The Luciferase plasmid was utilised
as a positive control for the reaction. The numbers on the left
indicate the position of molecular size markers for reference. The
size of the band denoting ECSM4 is consistent with the calculated
molecular weight of the polypeptide of 118 kDa.
[0445] FIG. 4.
[0446] cDNA and computer translation of GenBank AK000805 (human
ECSM4/magic roundabout).
[0447] FIG. 5.
[0448] Phrap generated contig sequence for human ECSM4 (magic
roundabout) ESTs and translation of the encoded polypeptide. The
DNA sequence is shown in the orientation as if it were a cDNA,
which is opposite to that in which it was originally generated. The
ESTs used to generate the contig are shown in Table 11. Translation
start in this sequence is at position 2 of the contig sequence, and
translation finish is at position 948.
[0449] FIG. 6.
[0450] An alignment of the GenBank Accession No AK000805
("magic.seq") and Phrap ("hs.111518") generated nucleic acid
sequences of human ECSM4 given in FIGS. 4 and 5.
[0451] FIG. 7.
[0452] Mouse ECSM4 contig nucleotide sequence and amino acid
sequence.
[0453] FIG. 8.
[0454] An alignment of the amino acid sequences of the mouse Robo1
protein ("T30805") and human ECSM4 ("magic.pep").
[0455] FIG. 9.
[0456] An alignment of the amino acid sequences of mouse Robo1
protein ("T30805") and mouse ECSM4 ("mousemagic.pep").
[0457] FIG. 10.
[0458] An alignment of the amino acid sequences of human
("magic.pep") and mouse ("mousemagic.pep") ECSM4 proteins. Residues
in bold indicate well conserved sequences. The mouse protein
sequence is shown on top and the human sequence is below.
[0459] FIG. 11.
[0460] Expression of magic roundabout in vitro. (a) Ribonuclease
protection analysis. Top, two probes to different regions
(nucleotides 1 to 355 and 3333 to 3679) of magic roundabout were
used in the analysis (shown left and right). RNase protection assay
was performed with U6 small nuclear RNA as control (shown bottom)
(Maxwell et al (1999) Nature 399: 271). Human cell lines and
primary isolates: MRC-5, fibroblast cell line, MCF-7, breast
carcinoma cell line, Neuro, SY-SH-5Y neuroblastoma cell line,
HUVEC, umbilical vein endothelial isolate, HDMEC, dermal
microvascular endothelial isolate and HMME2, mammary microvascular
endothelial cell line. N, normoxia, H, hypoxia, P, proliferating.
(b) Western analysis of cell lysates. A band at 110 kD corresponds
to MR and was stronger in cells exposed to hypoxia for 18 h. The
experiment was repeated twice with similar results. Immunoblotting
was carried out as described in Brown et al (2000) Cancer Res. 60:
6298. Polyclonal rabbit anti-sera was raised against the following
peptides coupled to keyhole limpet haemocyanin: amino acids 165-181
(LSQSPGAVPQALVAWRA) and 274-288 (DSVLTPEEVALCLEL) (anti-sera 1) or
peptides 311-320 (TYGYISVPTA) and 336-351 (KGGVLLCPPRPCLTPT)
(anti-sera 2). Both anti-sera gave identical results. For western
analysis, anti-sera was affinity purified on a "Hi-Trap
NHS-activated HP" column (Amersham) to which the peptides used to
raise anti-sera 1 were coupled.
[0461] FIG. 12.
[0462] Human ECSM4 full-length cDNA and encoded protein
sequence.
[0463] FIG. 13.
[0464] Mouse ECSM4 full-length cDNA (MuMR.seq) and encoded protein
sequence.
[0465] FIG. 14.
[0466] Alignment of human ECSM4 (top) and mouse ECSM4 (bottom)
amino acid sequences.
[0467] FIG. 15.
[0468] Alignment of human ECSM4 ("HuMR.seq"; top) and mouse ECSM4
("MuMR.seq"; bottom) cDNA sequences.
[0469] FIG. 16.
[0470] In situ hybridisation-analysis of human placental tissue
using ECSM4 as probe. A bright field view of 10.times.
magnification of thin section of placental tissue. The arrow
indicates a large blood vessel.
[0471] FIG. 17.
[0472] In situ hybridisation analysis of human placental tissue
using ECSM4 as probe. A higher magnification of the bright-field
view of thin section of placental tissue shown in FIG. 16,
focussing on the blood vessel. The arrow points to endothelial
cells lining the lumen of the vessel.
[0473] FIG. 18.
[0474] In situ hybridisation analysis of human placental tissue
using ECSM4 as probe. A higher magnification of the thin section of
placental tissue shown in FIG. 16, focussing on the blood vessel
and shown here in dark-field. The arrow depicts positive staining
of endothelial cells lining the lumen of the vessel.
[0475] FIG. 19.
[0476] In situ hybridisation analysis of colorectal liver
metastatic tissue using ECSM4 as probe. A bright-field view of a
section of colorectal liver metastatic tissue magnified with (A)
10.times. and (B) 20.times. objective. The area marked by the
boundary (encircling * A) depicts the normal liver tissue. The
arrow in (B) shows one of the blood vessels within the metastatic
tumour tissue.
[0477] FIG. 20.
[0478] In situ hybridisation analysis of colorectal liver
metastatic tissue using ECSM4 as a probe. This is a dark field view
of a section of colorectal liver metastatic tissue magnified with
(A) 10.times. and (B) 20.times. objective. The area marked by the
boundary (encircling *) depicts the normal liver tissue. The arrow
in (B) shows one of the blood vessels within the metastatic tumour
tissue corresponding to the vessel shown in FIG. 19B. Expression of
ECSM4 is restricted to endothelial cells of the tumour blood
vessels. Note that there is little expression in the surrounding
normal tissue (*).
[0479] FIG. 21.
[0480] Western Blot using the rabbit antibody MGO-5 as primary
antibody. Dilutions of the peptides ECSM4-derived peptides MR 165,
MR 311, MR 366 and the control polypeptide Bovine Serum Albumin
(BSA) were resolved by SDS polyacrylamide gel electrophoresis and
blotted onto Immobilon P membrane. The blot was probed with MGO-5
antibody and visualised using anti-rabbit antibody coupled with
alkaline phosphatase.
[0481] FIG. 22.
[0482] Immunostaining of frozen placental section. A frozen thin
section of human placenta was analysed by immunohistochemistry
without any primary antibody (negative control) and visualised
using anti-rabbit antibody coupled with alkaline phosphatase.
Little background staining is observed.
[0483] FIG. 23.
[0484] Immunostaining of frozen placental section. A frozen thin
section of human placenta was analysed by immunohistochemistry
using a primary antibody recognising von Willibrand Factor
(positive control), and visualised using an anti-rabbit secondary
antibody coupled with alkaline phosphatase. The arrows show high
levels of expression of vWF restricted to the vascular endothelial
cells.
[0485] FIG. 24.
[0486] Immunostaining of frozen placental section. A frozen thin
section of human placenta was analysed by immunohistochemistry
using MGO-5 (a rabbit polyclonal antibody raised against peptide MR
165) as the primary antibody, and visualised using anti-rabbit
secondary antibody coupled with alkaline phosphatase. The arrows
show high levels of expression of ECSM4 restricted to the vascular
endothelial cells. Note that the surrounding tissue shows little
staining. Comparison with FIGS. 22 and 23 shows that the expression
of ECSM4 colocalises with that of vWF, a known marker for vascular
endothelial cells.
[0487] FIG. 25.
[0488] Immunohistochemical analysis of HUVEC cells: von Willibrand
Factor (vWF). HUVEC cells were immobilised and analysed by
immunohistochemistry using an antibody recognising von Willibrand
Factor (a marker for endothelial cells) as the primary antibody and
visualised using anti-rabbit antibody coupled with alkaline
phosphatase. The arrows show expression of vWF in a subset of the
HUVEC cells.
[0489] FIG. 26.
[0490] Immunohistochemical analysis of HUVEC cells using the
antibody MGO-7. HUVEC cells were immobilised and analysed by
immunohistochemistry using MGO-7 antibody (a rabbit polyclonal
antibody raised against peptides MR 311 and MR 336) as the primary
antibody and visualised using anti-rabbit antibody coupled with
alkaline phosphatase. The arrows show expression of ECSM4 in a
subset of the HUVEC cells. Note that the staining is localised
primarily to the cell surface of the cells.
[0491] FIG. 27. Expression of magic roundabout in vivo.
[0492] (A) Expression of MR detected by in situ hybridisation in of
a placental arteriole (a) and venule (b) (left, light field and
right, dark field). (c) Immunohistochemical staining of magic
roundabout in a placental arteriole. Left, von Willibrand factor
control and right, magic roundabout. (B) Expression of MR in tumour
endothelium. Ganglioglioma (a) .times.20 and (b) .times.50. Left,
light field; right, dark field. Arrows highlight a vessel running
diagonally down the section with an erythrocyte within it.
Endothelial cells are strongly positive for MR expression.
Papillary bladder carcinoma (c) .times.20 and (d) .times.50. The
vascular core of the papilla of the tumour is strongly positive,
particularly the `flat` endothelial cells indicated by arrows. A
magic roundabout antisense in situ probe was generated using T3
polymerase from IMAGE EST clone 1912098 (GenBank acc. AI278949).
The plasmid was linearised with Eco RI prior to probe synthesis. In
situ analysis was then performed as described in Poulsom et al
(1998) Eur. J. Histochemistry 42:121-132.
EXAMPLE 1
In Silico Cloning of Novel Endothelial Specific Genes.
[0493] We describe the use of two independent strategies for
differential expression analysis combined with experimental
verification to identify genes specifically or preferentially
expressed in vascular endothelium.
[0494] The first strategy was based the EST cluster expression
analysis in the human UniGene gene index (Schuler et al, 1997).
Recurrent gapped BLAST searches (Altschul et al, 1997) were
performed at very high stringency against expressed sequence tags
(ESTs) grouped in two pools. These two pools comprised endothelial
cell and non-endothelial cell libraries derived from dbEST (Boguski
et al, 1995). The second strategy employed a second datamining
tool: SAGEmap xProfiler. XProfiler is a freely available on-line
tool, which is a part of the NCBI's Cancer Genome Anatomy Project
(CGAP) (Strausberg et al, 1997, Cole et al, 1995). While these two
approaches alone were producing a discouragingly high number of
false positives, when both strategies were combined, predictions
proved exceptionally reliable and two novel candidate
endothelial-specific genes have been identified. Full-length cDNAs
have been identified in sequence databases. Another gene (EST
cluster) corresponds to a partial cDNA sequence from a large-scale
cDNA sequencing project and contains a region of similarity to the
intracellular domain of human roundabout homologue 1 (ROBO1).
UniGene/EST Gene Index Screen
[0495] A pool of endothelial and a pool of non-endothelial
sequences were extracted using Sequence Retrieval System (SRS)
version 5 from dbEST. The endothelial pool consisted of 11,117 ESTs
from nine human endothelial libraries (Table 1). The
non-endothelial pool included 173,137 ESTs from 108 human cell
lines and microdissected tumour libraries (Table 2). ESTs were
extracted from dbEST release April 2000. Multiple FASTA files were
transformed into a BLAST searchable database using the pressdb
programme. Table 3 shows the expression status of five known
endothelial cell-specific genes in these two pools.
[0496] Subsequently, the longest, representative sequence in each
UniGene cluster (UniGene Build #111 May 2000, multiple FASTA file
hs.seq.uniq) was searched using very high stringency BLAST against
these two pools. If such representative sequence reported no hits,
the rest of the sequences belonging to the cluster (UniGene
multiple-FASTA file hs.seq) were used as BLAST queries. Finally,
clusters with no hits in the non-endothelial pool and at least one
hit in the endothelial pool were selected.
[0497] Optimising the BLAST E-value was crucial for the success of
BLAST identity-level searches. Too high an E-value would result in
gene patalogues being reported. On the other hand, too low
(stringent) an E-parameter would result in many false negatives,
i.e. true positives would not be reported due to sequencing errors
in EST data: ESTs are large-scale low-cost single pass sequences
and have high error rate (Aaronson et al, 1996). In this work an
E-value of 10e-20 was used in searches against non-endothelial EST
pool and a more stringent 10e-30 value in searches against the
smaller endothelial pool. These values were deemed optimal after a
series of test BLAST searches.
SAGE Data and SAGEmap xProfiler Differential Analysis
[0498] Web-based SAGE library subtraction (SAGEmap xProfiler:
http://www.ncbi.nlm.nih.gov/SAGE/sagexpsetup.cgi) was utilised as
the second datamining strategy for the identification of novel
endothelial specific or preferentially endothelial genes. Two
endothelial SAGE libraries (SAGE Duke_VEC and SAGE_Duke_HMVEC+VEGF
with a total of 110,790 sequences) were compared to twenty-four
non-endothelial, cell line libraries (full list in Table 4, total
of 733,461 sequences). Table 5 shows the status of expression of
five known endothelial specific genes: von Willebrand's factor
(vWF), two vascular endothelial growth factor receptors: fins-like
tyrosine kinase 1 (flt1) and kinase insert domain receptor (KDR),
tyrosine kinase receptor type tie (TIE1) and tyrosine kinase
receptor type tek (TIE2/TEK), in these two SAGE pools.
Combined Data Gives Highly Accurate Predictions
[0499] Twenty known genes were selected in the UniGene/EST screen
(Table 6). These genes had no hits in the non-endothelial pool and
at least one hit in the endothelial pool. The list contained at
least four endothelial specific genes: TIE1, TIE2/TEK, LYVE1 and
multimerin, indicating .about.20% accuracy of prediction. Other
genes on the list, while certainly preferentially expressed in the
endothelial cells, might not be endothelial specific. To improve on
the prediction accuracy we decided to combine UniGene/EST screen
with the xProfiler SAGE analysis. The xProfiler output consisted of
a list of genes with a ten times higher number of tags in the
endothelial than in the non-endothelial pool sorted according to
the certainty of prediction. A 90% certainty threshold was applied
to this list. Table 7 shows how data from the two approaches were
combined. Identity-level BLAST searches were performed on mRNAs
(known genes) or phrap computed contigs (EST clusters representing
novel genes) to investigate how these genes were represented in the
endothelial and non-endothelial pool. Subsequent experimental
verification by RT-PCR (FIG. 1) proved that the combined approach
was 100% accurate, i.e. genes on the xProfiler list which had no
matches the non-endothelial EST pool and at least one match in the
endothelial pool were indeed endothelial specific.
Discussion
[0500] There have been several reports of computer analysis of
tissue transcriptosomes. Usually an expression profile is
constructed, based on the number of tags assigned to a given gene
or a class of genes (Bernstein et al, 1996, Welle et al, 1999,
Bortoluzzi et al, 2000). An attempt can be made to identify
tissue-specific transcripts, for example Vasmatzis et al, (1997)
described three novel genes expressed exclusively in the prostate
by in silico subtraction of libraries from the dbEST collection.
Purpose made cDNA libraries may also be employed. Ten candidate
granulocyte-specific genes have been identified by extensive
sequence analysis of cDNA libraries derived from granulocytes and
eleven other tissue samples, namely a hepatocyte cell line, foetal
liver, infant liver, adult liver, subcutaneous fat, visceral fat,
lung, colonic mucosa, keratinocytes, cornea and retina (Itoh et al,
1998).
[0501] An analysis similar to the dbEST-based approach taken by
Vasmatzis et al, is complicated by the fact that endothelial cells
are present in all tissues of the body and endothelial-ESTs are
contaminating all bulk tissue libraries. To validate this we used
three well-known endothelial specific genes: KDR, FLT1, and TIE-2
as queries for BLAST searches against dbEST. Transcripts were
present in a wide range of tissues with multiple hits in well
vascularised tissues (e.g. placenta, retina), embryonic (liver,
spleen) or infant (brain) tissues. Additionally, we found that
simple subtraction of endothelial EST libraries against all other
dbEST libraries failed to identify any specific genes (data not
shown).
[0502] Two very different types of expression data resources were
used in our datamining efforts. The UniGene/EST screen was based on
expressed sequence tag libraries from dbEST. There are 9 human
endothelial libraries in the current release of dbEST with a
relatively small total number of ESTs: .about.11,117. Some
well-known endothelial specific genes are not represented in this
dataset (Table 3). This limitation raised our concerns that genes
with low levels of expression would be overlooked in our analysis.
Therefore, we utilised another type of computable expression data:
CGAP SAGE libraries. SAGE tags are sometimes called small ESTs
(usually 10-11 bp in length). Their major advantage is that they
can be unambiguously located within the cDNA: they are immediately
adjacent to the most 3' NlaIII restriction site. Though, there are
only two endothelial CGAP SAGE libraries available at the moment,
they contain an impressive total of .about.111,000 tags--an
approximately times bigger dataset than the .about.11,117 sequences
in the endothelial EST pool. The combined approach proved very
accurate (Table 8, FIG. 1) when verified by RT-PCR.
[0503] We report here identification of two novel highly
endothelial specific genes: endothelial cell-specific molecule 1
(ECSM1--UniGene entry Hs.13957) and magic roundabout (UniGene entry
Hs.111518). For a comprehensive summary of data available on these
genes see Table 8.
[0504] Our combined datamining approach together with experimental
verification is a powerful functional genomics tool. This type of
analysis can be applied to many cell types not just endothelial
cells. The challenge of identifying the function of discovered
genes remains, but bioinformatics tools such as structural
genomics, or homology and motif searches can offer insights that
can then be verified experimentally.
[0505] In summary, this screening approach has allowed the
identification of novel endothelial cell specific genes and known
genes whose expression was not known to be specific to endothelial
cells. This identification both advances our understanding of
endothelial cell biology and provides new pharmaceutical targets
for imaging, diagnosing and treating medical conditions involving
the endothelium.
Methods
PERL scripts
[0506] A number of PERL scripts were generated to facilitate large
scale sequence retrieval, BLAST search submissions, and automatic
BLAST output analysis.
Database Sequence Retrieval
[0507] Locally stored UniGene files (Build #111, release date May
2000) were used in the preparation of this report. The UniGene
website can be accessed on the URL: www.ncbi.nlm.nih.gov/UniGene/,
and UniGene files can be downloaded from the ftp repository:
ftp://ncbi.nlm.nih.gov/repository/unigene/. Representative
sequences for the human subset of UniGene (the longest EST within
the cluster) are stored in the file Hs.seq.uniq, while all ESTs
belonging to the cluster are stored in a separate file called
Hs.seq.
[0508] Sequences were extracted from the dbEST database accessed
locally at the HGMP centre using the Sequence Retrieval System (SRS
version 5) getz command. This was done repeatedly using a PERL
script for all the libraries in the endothelial and non-endothelial
subsets, and sequences were merged into two multiple-FASTA
files.
Selection Criteria for Non-Endothelial EST Libraries
[0509] Selection of 108 non-endothelial dbEST libraries was largely
manual. Initially the list of all available dbEST libraries
(http://www.ncbi.nlm.nih.gov/dbEST/libs_byorg.html) was searched
using the keyword `cells` and the phrase `cell line`. While this
searched identified most of the libraries, additional keywords had
to be added for the list to be full: `melanocyte`, `macrophage`,
`HeLa`, `fibroblast`. In some cases, detailed library description
was consulted to confirm that library is derived from a cell
line/primary culture. We also added a number of CGAP microdissected
tumour libraries. For that, Library Browser (available at
http://www.ncbi.nlm.nih.gov/CGAP/hTGI/Ibrow/cgaplb.cgi) was used to
search for the keyword `microdissected`.
UniGene Gene Index Screen
[0510] The UniGene gene transcript index was screened against the
EST division of GenBank, dbEST. Both UniGene and dbEST were
developed at the National Centre for Biotechnology Information
(NCBI). UniGene is a collection of EST clusters corresponding to
putative unique genes. It currently consists of four datasets:
human, mouse, rat and zebrafish. The human dataset is comprised of
approximately 90,000 clusters (UniGene Build #111 May 2000). By
means of very high stringency BLAST identity searches, we aimed to
identify those UniGene genes that have transcripts in the
endothelial and not in the non-endothelial cell-type dbEST
libraries. Throughout the project, University of Washington blast2
which is a gapped version was used as BLAST implementation. The
E-value was set to 10e-20 in searches against the non-endothelial
EST pool and to 10e-30 in searches against the smaller endothelial
pool.
[0511] While UniGene does not provide consensus sequences for its
clusters, the longest sequence within the cluster is identified.
Thus, this longest representative sequence (multiple FASTA file
Hs.seq.uniq) was searched using very high stringency BLAST against
the endothelial and non-endothelial EST pool. If such
representative sequence reported no matches, the rest of the
sequences belonging to the cluster (UniGene multiple-FASTA file
Hs.seq) followed as BLAST queries. Finally, clusters with no
matches in the non-endothelial pool and at least one match in the
endothelial pool were selected using PERL scripts analysing BLAST
textual output.
xProfiler SAGE Subtraction
[0512] xProfiler enables an on-line user to perform a differential
comparison of any combination of forty seven serial analysis of
gene expression (SAGE) libraries with a total of .about.2,300,000
SAGE tags using a dedicated statistical algorithm (Chen et al,
1998). xProfiler can be accessed on:
http://www.ncbi.nlm.nih.gov/SAGE/sagexpsetup.cgi. SAGE itself is a
quantitative expression technology in which genes are identified by
typically a 10 or 11 bp sequence tag adjacent to the cDNA's most 3'
NlaIII restriction site (Velculescu et al, 1995).
[0513] The two available endotbelial cell libraries
(SAGE_Duke_HMVEC and SAGE_Duke_HMVEC+VEGF) defined pool A and
twenty-four (see Table 4 for list) non-endothelial libraries
together built pool B. The approach was verified by establishing
the status of expression of the five reference endothelial specific
genes in the two SAGE pools (Table 5) using Gene to Tag Mapping
(http://www.ncbi.nlm.nih.gov/SAGE/SAGEcid.cgi). Subsequently,
xProfiler was used to select genes differentially expressed between
the pools A and B. The xProfiler output consisted of a list of
genes with a ten fold difference in the number of tags in the
endothelial compared to the non-endothelial pool sorted according
to the certainty of prediction. A 90% certainty threshold was
applied to this list.
[0514] The other CGAP's on-line differential expression analysis
tool, Digital Differential Display (DDD), relies on EST expression
data (source library info) instead of using SAGE tags. We attempted
to utilise this tool similarly to SAGEmap xProfiler but have been
unable to obtain useful results. Five out of nine endothelial and
sixty-four out of hundred and eight non-endothelial cell libraries
used in our BLAST-oriented approach were available for on-line
analysis using DDD (http://www.ncbi.nlm.nih.gov/CGAP/info/ddd.cgi).
When such analysis was performed the following were fifteen top
scoring genes: annexin A2, actin gamma 1, ribosomal protein large
P0, plasminogen activator inhibitor type I, thymosin beta 4,
peptidyloprolyl isomerase A, ribosomal protein L13a, laminin
receptor 1 (ribosomal protein SA), eukaryotic translation
elongation factor 1 alpha 1, vimentin, ferritin heavy polypeptide,
ribosomal protein L3, ribosomal protein S18, ribosomal protein L19,
tumour protein translationally-controlled 1. This list was rather
surprising, did not include any well-known endothelial specific
genes, did not have any overlap with SAGE results (Table 8), and
contained many genes, that in the literature are reported to be
ubiquitously expressed (ribosomal proteins, actin, vimentin,
ferritin). A major advantage of our UniGene/EST screen is that
instead of relying on source library data and fallible EST
clustering algorithms it actually performs identity-level BLAST
comparisons in search of transcripts corresponding to a gene.
Mining Data on UniGene Clusters
[0515] To quickly access information about UniGene entries (e.g.
literature references, STS sites, homologues, references to
function) on-line resources were routinely used: NCBI's UniGene and
LocusLink interfaces and Online Mendelian Inheritance in Man.
[0516] ESTs in UniGene clusters are not assembled into contigs, so
before any sequence analysis, contigs were created using phrap
assembler (for documentation on phrap see
http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).
[0517] To analyse genomic contig AC005795 (44,399) bp containing
ECSM1, NIX Internet interface for multi-application analysis of
large unknown nucleotide sequences was used. For further
information on NIX see http://www.hgmp.mrc.ac.uk/NIX/. Alignment of
ECSM1 against AC005795 was obtained using the NCBI interface to the
Human Genome Interface: the NCBI Map Viewer. For further
information on the NCBI Map Viewer see
http://www.ncbi.nlm.nih.gov/genome/guide/.
[0518] To search for possible transmembrane domains and signal
sequences in translated nucleotide sequences three Internet based
applications were used: DAS
http://www.biokemi.su.se/.about.server/DAS/(Cserzo et al, 1997),
TopPred2 http://www.biokemi.su.se/.about.server/toppred2/(Heijne
1992), and SignalP http://www.cbs.dtu.dk/services/SignalP/(Nielsen
et al, 1997).
PERL scripts
[0519] A number of PERL scripts were generated to facilitate large
scale sequence retrieval, BLAST search submissions, and automatic
BLAST output analysis.
Experimental Verification
[0520] To experimentally verify specificity of expression we used
the reverse transcription polymerase chain reaction (RT-PCR). RNA
was extracted from three endothelial and seven non-endothelial cell
types cultured in vitro. Endothelial cultures were as follows:
HMVEC (human microvascular endothelial cells), HUVEC (human
umbilical vein endothelial cells) confluent culture and HUVEC
proliferating culture. Non-endothelial cultures were as follows:
normal endometrial stromal (NES) cells grown in normoxia and NES
grown in hypoxia, MDA 453 and MDA 468 breast carcinoma cell lines,
HeLa, FEK4 fibroblasts cultured in normoxia and FEK4 fibroblasts
cultured in hypoxia, and SW480, HCT116--two colorectal epithelium
cell lines.
[0521] If a sequence tagged site (STS) was available, dbSTS PCR
primers were used and cycle conditions suggested in the dbSTS entry
followed. Otherwise, primers were designed using the Primer3
programme. Primers are listed in Table 9.
Tissue Culture Media, RNA Extraction and cDNA Synthesis
[0522] Cell-lines were cultured in vitro according to standard
tissue culture protocols. In particular, endothelial media were
supplemented with ECGS (endothelial cell growth supplement--Sigma),
and heparin (Sigma) to promote growth.
[0523] Total RNA was extracted using the RNeasy Minikit (Qiagen)
and cDNA synthesised using the Reverse-IT 1.sup.st Strand Synthesis
Kit (ABgene).
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TABLE-US-00002 TABLE 1 Nine human endothelial libraries from dbEST
Human aortic endothelium, 20 sequences, in vitro culture Human
endothelial cells, 346 sequences, primary isolate Human endothelial
cell (Y. Mitsui), 3 sequences, in vitro culture Stratagene
endothelial cell 937223, 7171 sequences, primary isolate Aorta
endothelial cells, 1245 sequences, primary isolate Aorta
endothelial cells, TNF treated, 1908 sequences, primary isolate
Umbilical vein endothelial cells I, 9 sequences HDMEC cDNA library,
11 sequences, in vitro culture Umbilical vein endothelial cells II,
404 sequences
[0576] TABLE-US-00003 TABLE 2 Non-endothelial dbEST libraries. 1.
Activated T-cells I 2. Activated T-cells II 3. Activated T-cells
III 4. Activated T-cells IV 5. Activated T-cells IX 6. Activated
T-cells V 7. Activated T-cells VI 8. Activated T-cells VII 9.
Activated T-cells VIII 10. Activated T-cells X 11. Activated
T-cells XI 12. Activated T-cells XII 13. Activated T-cells XX 14.
CAMA1Ee cell line I 15. CAMA1Ee cell line II 16. CCRF-CEM cells,
cyclohexamide treated I 17. CdnA library of activated B cell line
3D5 18. Chromosome 7 HeLa cDNA Library 19. Colon carcinoma (Caco-2)
cell line I 20. Colon carcinoma (Caco-2) cell line II 21. Colon
carcinoma (HCC) cell line 22. Colon carcinoma (HCC) cell line II
23. HCC cell line (matastasis to liver in mouse) 24. HCC cell line
(matastasis to liver in mouse) II 25. HeLa cDNA (T. Noma) 26. HeLa
SRIG (Synthetic retinoids induced genes) 27. Homo sapiens
monocyte-derived macrophages 28. HSC172 cells I 29. HSC172 cells II
30. Human 23132 gastric carcinoma cell line 31. Human breast cancer
cell line Bcap 37 32. Human cell line A431 subclone 33. Human cell
line AGZY-83a 34. Human cell line PCI-O6A 35. Human cell line
PCI-O6B 36. Human cell line SK-N-MC 37. Human cell line TF-1
(D.L.Ma) 38. Human exocervical cells (CGLee) 39. Human fibrosarcoma
cell line HT1080 40. Human fibrosarcoma cell line HT1080-6TGc5 41.
Human gastric cancer SGC-7901 cell line 42. Human GM-CSF-deprived
TF-1 cell line (Liu, Hongtao) 43. Human HeLa (Y. Wang) 44. Human
HeLa cells (M. Lovett) 45. Human Jurkat cell line mRNA (Thiele, K.)
46. Human K562 erythroleukemic cells 47. Human lung cancer cell
line A549.A549 48. Human nasopharyngeal carcinoma cell line HNE1
49. Human neuroblastoma SK-ER3 cells (M. Garnier) 50. Human newborn
melanocytes (T. Vogt) 51. Human pancreatic cancer cell line Patu
8988t 52. Human primary melanocytes mRNA (I. M. Eisenbarth) 53.
Human promyelocytic HL60 cell line (S. Herblot) 54. Human retina
cell line ARPE-19 55. Human salivary gland cell line HSG 56. Human
White blood cells 57. Jurkat T-cells I 58. Jurkat T-cells II 59.
Jurkat T-cells III 60. Jurkat T-cells V 61. Jurkat T-cells VI 62.
Liver HepG2 cell line. 63. LNCAP cells I 64. Macrophage I 65.
Macrophage II 66. Macrophage, subtracted (total CdNA) 67. MCF7 cell
line 68. Namalwa B cells I 69. Namalwa B cells II 70. NCI_CGAP_Br4
71. NCI_CGAP_Br5 72. NCI_CGAP_CLL1 73. NCI_CGAP_GCB0 74.
NCI_CGAP_GCB1 75. NCI_CGAP_HN1 76. NCI_CGAP_HN3 77. NCI_CGAP_HN4
78. NCI_CGAP_HSC1 79. NCI_CGAP_Li1 80. NCI_CGAP_Li2 81.
NCI_CGAP_Ov5 82. NCI_CGAP_Ov6 83. NCI_CGAP_Pr1 84. NCI_CGAP_Pr10
85. NCI_CGAP_Pr11 86. NCI_CGAP_Pr16 87. NCI_CGAP_Pr18 88.
NCI_CGAP_Pr2 89. NCI_CGAP_Pr20 90. NCI_CGAP_Pr24 91. NCI_CGAP_Pr25
92. NCI_CGAP_Pr3 93. NCI_CGAP_Pr4 94. NCI_CGAP_Pr4.1 95.
NCI_CGAP_Pr5 96. NCI_CGAP_Pr6 97. NCI_CGAP_Pr7 98. NCI_CGAP_Pr8 99.
NCI_CGAP_Pr9 100. Normal Human Trabecular Bone Cells 101. Raji
cells, cyclohexamide treated I 102. Retinal pigment epithelium 0041
cell line 103. Retinoid treated HeLa cells 104. Soares melanocyte
2NbHM 105. Soares_senescent_fibroblasts_Nb HSF 106. Stratagene HeLa
cell s3 937216 107. Supt cells 108. T, Human adult Rhabdomyosarcoma
cell-line
[0577] TABLE-US-00004 TABLE 3 Five genes known to be endothelial
specific genes in the dbEST pools. The number of ESTs in the
endothelial pool is relatively small (.about.11,117) and not all
known endothelial genes are represented Known endothelial specific
Hits in the non- Hits in the gene endothelial pool endothelial pool
von Willebrand factor (vWF) 1 27 flt1 VEGF receptor -- -- KDR VEGF
receptor 1 -- TIE1 tyrosine kinase -- 5 TIE2/TEK tyrosine kinase --
2
[0578] TABLE-US-00005 TABLE 4 Twenty-four non-endothelial cell
SAGE-CGAP libraries. SYMBOL DESCRIPTION SAGE_HCT116 Colon, cell
line derived from colorectal carcinoma SAGE_Caco_2 Colon,
colorectal carcinoma cell line SAGE_Duke_H392 Brain, Duke
glioblastoma multiforme cell line SAGE_SW837 Colon, cancer cell
line SAGE_RKO Colon, cancer cell line SAGE_NHA(5th) Brain, normal
human astrocyte cells harvested at passage 5 SAGE_ES2-1 Ovarian
Clear cell carcinoma cell line ES-2, poorly differentiated
SAGE_OVCA432-2 Ovary, carcinoma cell line OVCA432 SAGE_OV1063-3
Ovary, carcinoma cell line OV1063 SAGE_Duke_mhh-1 Brain, c-myc
negative medulloblastoma cell line mhh-1 SAGE_Duke_H341 Brain,
c-myc positive medulloblastoma cell line H341 SAGE_HOSE_4 Ovary,
normal surface epithelium SAGE_OVP-5 Ovary, pooled cancer cell
lines SAGE_LNCaP Prostate, cell line. Androgen dependent
SAGE_HMEC-B41 Cell culture HMEC-B41 of normal human mammary
epithelial cells SAGE_MDA453 Cell line MDA-MB-453 of human breast
carcinoma SAGE_SKBR3 ATCC cell line SK-BR-3. Human breast
adenocarcinoma SAGE_A2780-9 Ovary, ovarian cancer cell line A2780
SAGE_Duke_H247_normal Brain, glioblastoma multiforme cell line,
H247 AGE_Duke_H247_Hypoxia Brain, Duke glioblastoma multiforme cell
line, H247, grown under 1.5% oxygen
SAGE_Duke_post_crisis_fibroblasts Skin, post-crisis survival
fibroblast cell-line SAGE_Duke_precrisis_fibroblasts Skin, large T
antigen transformed human fibroblasts clones SAGE_A Prostate,
cancer cell line. Induced with synthetic androgen SAGE_IOSE29-11
Ovary, surface epithelium line
[0579] TABLE-US-00006 TABLE 5 Five known endothelial specific genes
in the CGAP SAGE pools. TIE1 and TIE2/TEK have multiple hits in the
non-endothelial pool (most in normal or carcinoma cell lines of
ovarian origin). vWF is most endothelial specific having 80 hits in
the endothelial pool and only one hit in the non-endothelial pool.
Tags in the Known endothelial Tags in the non-endothelial
endothelial sage specific gene sage libraries libraries von
Willebrand factor 1 (colon carcinoma cell line) 80 (VWF) flt1 VEGF
receptor -- -- KDR VEGF receptor 1 (IOSE29 ovarian surface 6
epithelium cell line) TIE1 tyrosine kinase 17 (ovarian tumour and
27 normal ovarian epithelium cell lines) TIE2/TEK tyrosine 4
(ovarian carcinoma and 2 kinase glioblastoma multiforme cell
lines)
[0580] TABLE-US-00007 TABLE 6 Results of the UniGene/EST screen.
Twenty known genes were selected in the UniGene/EST screen (no hits
in the non-endothelial pool and minimum one hit in the endothelial
pool). At least four of these genes are known endothelial specific
genes: TIE1, TIE2/TEK, LYVE1 and multimerin, indicating .about.20%
prediction accuracy. Other genes, while certainly preferentially
expressed in the endothelial cells, may not be endothelial
specific. UniGene Endothelial Description ID hits TIE1 receptor
endothelial tyrosine kinase Hs.78824 5 Cytosolic phospholipase A2;
involved in the Hs.211587 3 metabolism of eicosanoids Branched
chain alpha-ketoacid dehydrogenase Hs.1265 2 CGMP-dependent protein
kinase; cloned from Hs.2689 2 aorta cDNA, strongly expressed in
well vascularised tissues like aorta, heart, and uterus (Tamura et
al, 1996) Lymphatic vessel endothelial hyaluronan Hs.17917 2
receptor 1 - LYVE1 (Banerji et al, 1999) TRAF interacting protein:
TNF signalling Hs.21254 2 pathway Multimerin: a very big
endothelial specific Hs.32934 2 protein; binds platelet factor V,
can also be found in platelets (Hayward et al, 1996) Diubiquitin (a
member of the ubiquitin family); Hs.44532 2 reported in dendrytic
and B lymphocyte cells; involved in antigen processing; this is
first evidence that it is also present in endothelial cells (Bates
et al, 1997) Beta-transducin family protein; also a homolog
Hs.85570 2 of D. melanogaster gene notchless: a novel WD40 repeat
containing protein that modulates Notch signalling activity
TIE2/TEK receptor endothelial tyrosine kinase Hs.89640 2 BCL2
associated X protein (BAX) Hs.159428 2 Sepiapterin reductase mRNA
Hs.160100 2 Retinoic acid receptor beta (RARB) Hs.171495 2 ST2
receptor: a homolog of the interleukin 1 Hs.66 1 receptor Mitogen
activated protein kinase 8 (MAPK8) Hs.859 1 ERG gene related to the
ETS oncogene Hs.45514 1 PP35 similar to E. coli yhdg and R.
Capsulatus Hs.97627 1 nifR3 Interphotoreceptor matrix proteoglycan;
Hs.129882 1 strongly expressed in retina and umbilical cord vein
(Felbor et al, 1998) Methylmalonate semialdehyde dehydrogenase
Hs.170008 1 gene, HTLV-I related endogenous retroviral sequence
Hs.247963 1
[0581] TABLE-US-00008 TABLE 7 xProfiler differential analysis was
combined with data from the UniGene/EST screen achieving 100%
certainty of prediction. xProfiler's output lists genes with
10-times higher number of tags in the endothelial than in the
non-endothelial pool of SAGE-CGAP libraries. Hits corresponding to
these genes in the endothelial and non-endothelial EST pools were
identified by identity-level BLAST searches for mRNA (known genes)
or phrap computed contig sequences (EST clusters representing novel
genes). Genes are sorted according to the number of hits in the
non-endothelial EST pool. Known and predicted novel endothelial
specific genes are in bold. Hits in X profiler Hits in non- Unigene
prediction endothelial endothelial ID Gene description certainty
EST pool EST pool Hs.13957 ESTs - ECSM1 97% 4 0 Hs.111518 magic
roundabout, 100% 4 0 distant homology to human roundabout 1
Hs.268107 multimerin 92% 5 0 Hs.155106 calcitonin receptor- 97% 0 0
like receptor activity modifying protein 2 Hs.233955 ESTs 96% 0 0
Hs.26530 serum deprivation 94% 3 1 response (phosphatidylserine-
binding protein) Hs.83213 fatty acid binding 100% 3 1 protein 4
Hs.110802 von Willebrand 100% 25 1 factor Hs.76206 cadherin 5, VE-
100% 4 1 cadherin (vascular endothelium) Hs.2271 endothelin 1 98% 9
2 Hs.119129 collagen, type IV, 100% 4 6 alpha 1 Hs.78146
platelet/endothelial 99% 18 5 cell adhesion molecule (CD31 antigen)
Hs.76224 EGF-containing 100% 37 9 fibulin-like extracellular matrix
protein 1 Hs.75511 connective tissue 100% 34 48 growth factor
[0582] TABLE-US-00009 TABLE 8 Summary of available information on
magic roundabout. UniGene Transmembrane Mapping information cluster
ID Full-length Longest segments, Genomic context and size cDNA ORF
signal peptide Genomic clones Description ECSM1 Hs.13957 103 aa
Genomic neighbour: Tropomyosin 1100 bp confirmed dbSTS G26129 and
G28043 with Chr. 19 Gene Map 98: Marker 5'RACE SGC33470, Marker
stSG3414, IntervalD19S425-D19S418 AC005945, AC005795 (partial
identity) Magic Hs.111518 Partial 417 aa One transmembrane Genomic
neighbour: integral 468 aa region of homology to the roundabout
cDNA domain predicted by transmembrane protein 1 (ITM1) cytoplasmic
portion of the roundabout FLJ20798 TopPred2 and DAS. dbSTS G14646
and G14937 axon guidance protein family: human fis, clone No signal
peptide Chr. 11, Gene Map 98: Marker ROBO1, rat ROBO1 and mouse
dutt1 2076 bp ADSU02031 detected in the SHGC-11739, Interval (E =
1.3e-09) (acc. available 417 aa D11S1353-D11S93 ORF has no apparent
up-stream limit. AK000805) ORF (SignalP) This and size comparison
to ROBO1 1496 bp however the true (1651 aa) suggests that true
protein is protein product is very likely to be much larger very
likely to be Possible alternative polyA sites: the larger cDNA
clone from adipocyte tissue seems to be polyadenylated in a
different position to the sequence from the UniGene contig
[0583] TABLE-US-00010 TABLE 9 List of primers used in RT-PCR
reactions. dbSTS primers were used if a UniGene entry contained a
sequence tagged site (STS). Otherwise, primers were designed using
the Primer3 programme. Primers (sequence or Gene GenBank Accession
for the STS) ECSM1 - Hs.13957 G26129 Magic roundabout - Hs.111518
G14937 calcitonin receptor-like receptor G26129 activity modifying
2 Hs.233955 G21261 fatty acid binding protein 4 5'-TGC AGC TTC CTT
CTC ACC TT-3' 5'-TCA CAT CCC CAT TCA CAC TG-3' von Willebrand
factor 5'-TGT ACC ATG AGG TTC TCA ATG C-3' 5'-TTA TTG TGG GCT CAG
AAG GG-3' serum deprivation response protein G21528 collagen, type
IV, alpha 1 G07125 EGF-containing fibulin-like G06992 extracellular
matrix protein 1 connective tissue growth factor 5'-CAA ATG CTT CCA
GGT GAA AAA-3' 5'-CGT TCA AAG CAT GAA ATG GA-3'
[0584] TABLE-US-00011 TABLE 10 ESTs belonging to ECSM1 contig
sequence are as follows: EST SEQUENCES(30) AI540508,
cDNAcloneIMAGE: 2209821, Uterus, 3'read, 2.1 kb AI870175,
cDNAcloneIMAGE: 2424998, Uterus, 3'read, 1.7 kb AI978643,
cDNAcloneIMAGE: 2491824, Uterus, 3'read, 1.3 kb AI473856,
cDNAcloneIMAGE: 2044374, Lymph, 3'read AI037900, cDNAcloneIMAGE:
1657707, Wholeembryo, 3'read, 1.2 kb AI417620, cDNAcloneIMAGE:
2115082, 3'read, 1.0 kb AA147817, cDNAcloneIMAGE: 590062, 3'read
AA968592, cDNAcloneIMAGE: 1578323, 3'read, 0.7 kb AW474729,
cDNAcloneIMAGE: 2853635, Uterus, 3'read R02352, cDNAcloneIMAGE:
124282, 3'read, 0.7 kb R01889, cDNAcloneIMAGE: 124485, 5'read, 0.7
kb AA446606, cDNAcloneIMAGE: 783693, Wholeembryo, 3'read R02456,
cDNAcloneIMAGE: 124282, 5'read, 0.7 kb T72705, cDNAcloneIMAGE:
108686, 5'read, 0.7 kb R01890, cDNAcloneIMAGE: 124485, 3'read, 0.7
kb AA147925, cDNAcloneIMAGE: 590014, 5'read AI131471,
cDNAcloneIMAGE: 1709098, Heart 3'read, 0.6 kb AA733177,
cDNAclone399421, Heart, 3'read AI039489, cDNAcloneIMAGE: 1658903,
Wholeembryo, 3'read, 0.6 kb AI128585, cDNAcloneIMAGE: 1691245,
Heart, 3'read, 0.6 kb AI540506, cDNAcloneIMAGE: 2209817, Uterus,
3'read, 0.6 kb AA894832, cDNAcloneIMAGE: 1502815, Kidney, 3'read,
0.5 kb AW057578, cDNAcloneIMAGE: 2553014, Pooled, 3'read, 0.3 kb
AA729975, cDNAcloneIMAGE: 1257976, GermCell, 0.3 kb AI131016,
cDNAcloneIMAGE: 1706622, Heart, 3'read, 0.2 kb AA147965,
cDNAcloneIMAGE: 590062, 5'read AA446735, cDNAcloneIMAGE: 783693,
Wholeembryo, 5'read AA147867, cDNAcloneIMAGE: 590014, 3'read
AI497866, cDNAcloneIMAGE: 2125892, Pooled, 3'read T72636,
cDNAcloneIMAGE: 108686, 3'read, 0.7 kb
[0585] TABLE-US-00012 TABLE 11 ESTs within the magic roundabout
sequence: EST sequences in magic roundabout (55): AI803963,
cDNAcloneIMAGE: 2069520, 3'read, 0.9 kb W88669, cDNAcloneIMAGE:
417844, 3'read, 0.7 kb AI184863, cDNAcloneIMAGE: 1565500, Pooled,
3'read, 0.6 kb AA011319, cDNAcloneIMAGE: 359779, Heart, 3'read, 0.6
kb AA302765, cDNAcloneATCC: 194652, Adipose, 3'read AI278949,
cDNAcloneIMAGE: 1912098, Colon, 3'read, 0.7 kb AI265775,
cDNAcloneIMAGE: 2006542, Ovary, 3'read AA746200, cDNAcloneIMAGE:
1324396, Kidney, 0.5 kb N78762, cDNAcloneIMAGE: 301290, Lung,
3'read AI352263, cDNAcloneIMAGE: 1940638, Wholeembryo, 3'read, 0.6
kb AA630260, cDNAcloneIMAGE: 854855, Lung, 3'read, 0.5 kb C20950,
cDNAclone(no-name), 3'read W88875, cDNAcloneIMAGE: 417844, 5'read,
0.7 kb AA156022, cDNAcloneIMAGE: 590120, 3'read N93972,
cDNAcloneIMAGE: 309369, Lung, 3'read, 1.7 kb AI217602,
cDNAcloneIMAGE: 1732380, Heart, 3'read, 0.5 kb AW294276,
cDNAcloneIMAGE: 2726'347, 3'read AA010931, cDNAcloneIMAGE: 359779,
Heart, 5'read, 0.6 kb AA303624, cDNAcloneATCC: 115215, Aorta,
5'read AI366745, cDNAcloneIMAGE: 1935056, 3'read, 0.5 kb AA327257,
cDNAcloneATCC: 127927, Colon, 5'read C06489, cDNAclonehbc5849,
Pancreas BE218677, cDNAcloneIMAGE: 3176164, lung, 3'read AA335675,
cDNAcloneATCC: 137498, Testis, 5'read R84975, cDNAcloneIMAGE:
180552, Brain, 3'read, 2.1 kb AI926445, cDNAcloneIMAGE: 2459442,
Stomach, 3'read, 1.9 kb H61208, cDNAcloneIMAGE: 236318, Ovary,
3'read, 1.9 kb AA335358, cDNAcloneATCC: 137019, Testis, 5'read
AI129190, cDNAcloneIMAGE: 1509564, Pooled, 3'read, 0.8 kb T59188,
cDNAcloneIMAGE: 74634, Spleen, 5'read, 0.8 kb T59150,
cDNAcloneIMAGE: 74634, Spleen, 3'read, 0.8 kb R53174,
cDNAcloneIMAGE: 154350, Breast, 5'read, 0.8 kb AA156150,
cDNAcloneIMAGE: 590120, 5'read AA302509, cDNAcloneATCC: 114727,
Aorta, 5'read R99429, cDNAcloneIMAGE: 201985, 5'read, 2.4 kb
AI813787, cDNAcloneIMAGE: 2421627, Pancreas, 3'read, 1.2 kb H62113,
cDNAcloneIMAGE: 236316, Ovary, 5'read, 1.0 kb R16422,
cDNAcloneIMAGE: 129313, 5'read, 0.7 kb T48993, cDNActoneIMAGE:
70531, Placenta, 5'read, 0.6 kb T05694, cDNAcloneHFBDF13, Brain
R84531, cDNAcloneIMAGE: 180104, Brain, 5'read, 2.2 kb AI903080,
cDNAclone(no-name), breast AI903083, cDNAclone(no-name), breast
AA302764, cDNAcloneATCC: 194652, Adipose, 5'read AA341407,
cDNAcloneATCC: 143064, Kidney, 5'read W16503, cDNAcloneIMAGE:
301194, Lung, 5'read AW801246, cDNAclone(no-name), uterus AW959183,
cDNAclone(no-name) R85924, cDNAcloneIMAGE: 180104, Brain, 3'read,
2.2 kb AA358843, cDNAcloneATCC: 162953, Lung, 5'read BE161769,
cDNAclone(no-name), head-neck W40341, cDNAcloneIMAGE: 309369, Lung,
5'read, 1.7 kb AA876225, cDNAcloneIMAGE: 1257188, GermCell, 3'read
R99441, cDNAcloneIMAGE: 202009, 5'read, 2.3 kb W76132,
cDNAcloneIMAGE: 344982, Heart, 5'read, 1.4 kb,
[0586] TABLE-US-00013 TABLE 12 110 ESTs in the mouse magic
roundabout cluster (Mm.27782) AI427548, cDNAcloneIMAGE: 521115,
Muscle, 3'read AV022394, cDNAclone1190026N09, 3'read BB219221,
cDNAcloneA530053H04, 3'read AI604803, cDNAcloneIMAGE: 388336,
Embryo, 3'read AI504730, cDNAcloneIMAGE: 964027, Mammarygland,
3'read AI430395, cDNAcloneIMAGE: 388336, Embryo, 5'read AI181963,
cDNAcloneIMAGE: 1451626, Liver, 3'read AV020471,
cDNAclone1190017N14, 3'read BB219225, cDNAcloneA530053H12, 3'read
BB224304, cDNAcloneA530086A21, 3'read BB527740,
cDNAcloneD930042M18, 3'read W66614, cDNAcloneIMAGE: 388336, Embryo,
5'read BB097630, cDNAclone9430060E21, 3'read AI152731,
cDNAcloneIMAGE: 1478154, Uterus, 5'read AW742708, cDNAcloneIMAGE:
2780289, innerear, 170pooled, 3'read BB118169, cDNAclone9530064M17,
3'read AI839154, cDNAcloneUI-M-AO0-ach-e-11-0-UI, 3'read BB206388,
cDNAcloneA430075J10, 3'read BB381670, cDNAcloneC230015E01, 3'read
BB199721, cDNAcloneA430017A19, 3'read AI593217, cDNAcloneIMAGE:
1177959, Mammarygland, 3'read BB219411, cDNAcloneA530054L01, 3'read
BB220744, cDNAcloneA530061M19, 3'read BB220944,
cDNAcloneA530062O22, 3'read BB390078, cDNAcloneC230066L23, 3'read
BB220730, cDNAcloneA530061L13, 3'read AI615527, cDNAcloneIMAGE:
964027, Mammarygland, 5'read AI882477, cDNAcloneIMAGE: 1396822,
Mammarygland, 5'read AV025281, cDNAclone1200012D01, 3'read
BB470462, cDNAcloneD230033L23, 3'read BB247620,
cDNAcloneA730020G03, 3'read BB555377, cDNAcloneE330019B13, 3'read
BB512960, cDNAcloneD730043I21 BB400157, cDNAcloneC330017F17, 3'read
BB320465, cDNAcloneB230385O10, 3'read BB105670,
cDNAclone9430096H10, 3'read BB441462, cDNAcloneD030027B11, 3'read
BB137530, cDNAclone9830142O07, 3'read AA553155, cDNAcloneIMAGE:
964027, Mammarygland, 5'read BB319763, cDNAcloneB230382G07, 3'read
BB451051, cDNAcloneD130007I05, 3'read BB504672,
cDNAcloneD630049J11, 3'read AI429453, cDNAcloneIMAGE: 569122,
Embryo, 3'read BB190585, cDNAcloneA330062J23, 3'read BB257082,
cDNAcloneA730076M18, 3'read BB386699, cDNAcloneC230047P06, 3'read
BB295814, cDNAcloneB130042A09, 3'read BB450972,
cDNAcloneD130007A22, 3'read AA718562, cDNAcloneIMAGE: 1177959,
Mammarygland, 5'read BB223775, cDNAcloneA530083K18, 3'read
AV020555, cDNAclone1190018G05, 3'read BB226083,
cDNAcloneA530095K11, 3'read BB482105, cDNAcloneD430007O19, 3'read
BB381671, cDNAcloneC230015E02, 3'read BB383758,
cDNAcloneC230030C02, 3'read BB257519, cDNAcloneA730080D13, 3'read
BB265667, cDNAcloneA830021I17, 3'read BB254777,
cDNAcloneA730063K20, 3'read AV240775, cDNAclone4732443F15, 3'read
BB315010, cDNAcloneB230352H04, 3'read BB390074,
cDNAcloneC230066L16, 3'read BB517605, cDNAcloneD830025B17, 3'read
BB484410, cDNAcloneD430025H01, 3'read BB357583,
cDNAcloneC030022J01, 3'read AV225639, cDNAclone3830431D12, 3'read
BB554921, cDNAcloneE330016A12, 3'read BB161650,
cDNAcloneA130061H21, 3'read BB106720, cDNAclone9530002M22, 3'read
BB535465, cDNAcloneE030043P14, 3'read BB357738,
cDNAcloneC030024B10, 3'read AV285588, cDNAclone5031411M12 BB188339,
cDNAcloneA330048H22, 3'read AV337749, cDNAclone6430404F19, 3'read
BB065281, cDNAclone8030443H10, 3'read BB148059,
cDNAclone9930104N19, 3'read AV252251, cDNAclone4833438P20, 3'read
BB184506, cDNAcloneA330012J24, 3'read BB522445,
cDNAcloneD930007M08, 3'read BB520366, cDNAcloneD830041K23, 3'read
AV127290, cDNAclone2700047J01, 3'read BB248651,
cDNAcloneA730027F04, 3'read BB008452, cDNAclone4732482M24, 3'read
BB550719, cDNAcloneE230024C07, 3'read BB182033,
cDNAcloneA230095N14, 3'read BB480258, cDNAcloneD330045D17, 3'read
BB004855, cDNAclone4732463E03, 3'read AV379748,
cDNAclone9230013A19, 3'read BB552137, cDNAcloneE230035B12, 3'read
BB288263, cDNAcloneIMAGE: 3490042, mammary, 5'read BB215681,
cDNAcloneA530026M11, 3'read BB251356, cDNAcloneA730046B16, 3'read
BB503441, cDNAcloneD630043F10, 3'read BB500571,
cDNAcloneD630029E03, 3'read BB199833, cDNAcloneA430017K13, 3'read
BB533549, cDNAcloneE030030K03, 3'read BB098399,
cDNAclone9430063L18, 3'read BB213310, cDNAcloneA530009E09, 3'read
BB240699, cDNAcloneA630083B14, 3'read BB217106,
cDNAcloneA530040N24, 3'read BB057432, cDNAclone7120459H22, 3'read
BB214645, cDNAcloneA530021N22, 3'read BB218254,
cDNAcloneA530048K12, 3'read BB319841, cDNAcloneB230382O06, 3'read
BB459759, cDNAcloneD130063G22, 3'reed BB485618,
cDNAcloneD430032M09, 3'read BB517699, cDNAcloneD830025J18, 3'read
BB535595, cDNAcloneE030044M09, 3'read BB536291,
cDNAcloneE030049D17, 3'read BB552689, cDNAcloneE330001A16, 3'read
BB552709, cDNAcloneE33C001C16, 3'read
EXAMPLE 2
ECSM4 Expression is Restricted to Endothelial Cells.
[0587] In situ hybridisation (ISH) of tumour and normal tissues
showed that the expression of ECSM4 is restricted to vascular
endothelial cells in adult angiogenic vessels only. Analysis of
normal tissues showed that expression of ECSM4 is detected in human
placenta and umbilical cord foetal tissue 10.8 weeks menstrual age.
As shown in FIG. 16, ECSM4 expression is highly specific for the
vascular endothelial cells of the blood vessel in placenta.
Furthermore, expression was absent throughout a number of other
normal tissues that were analysed, including adult liver, brain
cerebrum and large vessels, prostate, colon, small bowel, heart,
eye (choroid and sclera), ovary, stomach, breast and foetal
bladder, testis, kidney (15.8 weeks) and foetal heart, kidney,
adrenal, intestine (11.3 weeks) foetal brain (10.6 weeks) and
foetal eye (16.5 weeks) (data not shown).
[0588] ISH analysis of colorectal liver metastasis biopsies showed
that expression of ECSM4 was restricted to vascular endothelial
cells of the tumour vessels only (FIGS. 17 and 18). No expression
was detected in the surrounding normal tissue. Furthermore the
enhanced expression in the vicinity of the necrotic tissues (FIG.
18, necrotic tissue is indicated by the bright signal labelled *)
is indicative and consistent with induction of ECSM4 expression by
hypoxia. As such, ECSM4 may be a novel hypoxia regulated gene.
[0589] The highly restricted expression pattern of ECSM4 in
angiogenic vessels in normal and tumour tissues in adult is
entirely consistent with the endothelial cell selective pattern of
expression determined by the in silico analysis described in
Example 1.
Methods
[0590] Blocks of formalin-fixed, paraffin-embedded tissues and
tumours were obtained from the archives of the Imperial Cancer
Research Fund Breast Pathology Group at Guys Hospital, London, UK.
An antisense riboprobe to ECSM4 cDNA was prepared for specific
localisation of the ECSM4 mRNA by in situ hybridisation. The
methods for pretreatment, hybridisation, washing, and dipping of
slides in Ilford K5 for autoradiography has been described
previously (Poulsom, R., Longcroft, J. M., Jeffrey, R. E., Rogers,
L., and Steel, J. H. (1998) Eur. J. Histochem. 42, 121-132). Films
were exposed for 7 to 15 days before developing in Kodak D19 and
counterstaining with Giemsa. Sections were examined under
conventional or reflected light dark-field conditions (Olympus BH2
with epi-illumination) under a .times.5, .times.10 or .times.20
objective that allowed individual auto-radiographic silver grains
to be seen as bright objects on a dark background.
EXAMPLE 3
ECSM4 Polypeptide is Detected only in Endothelial Cells.
[0591] Antibodies capable of selectively binding the ECSM4
polypeptide were generated and used in immunohistochemistry to
demonstrate the presence of ECSM4 polypeptide in a range of cell
types (FIGS. 21 to 26). Tissue samples were prepared by standard
techniques in the art of immunohistochemistry.
Generation of Antibodies Recognising ECSM4.
[0592] The peptides MR 165, MR 311 and MR 336 were fused to Keyhole
Limpet Haemocyanin (KLH) before immunisation of rabbits for
production of polyclonal antibodies. The antibody MGO-5 was derived
from rabbits immunised with the peptide MR 165, whereas MGO-7 was
derived from rabbits immunised with a mixture of MR 311 and MR 336.
The sequence of the peptides used to generated the polyclonal
antibodies is shown below with their reference within the amino
acid sequence of full length human ECSM4 as shown in FIG. 12.
TABLE-US-00014 MR 165 = LSQSPGAVPQALVAWRA (681-697) MR 274 =
DSVLTPEEVALCLEL (790-804) MR 311 = TYGYISVPTA (827-836) MR 336 =
KGGVLLCPPRPCLTPT (852-867)
EXAMPLE 4
[0593] The magic roundabout EST sequence identified in the
bioinformatics search for endothelial specific transcripts was used
to isolate a cDNA of 3800 base pairs in length from a human heart
cDNA library. A screen using gene specific primers showed the gene
to be present in libraries from heart, adult and foetal brain,
liver, lung, kidney, muscle, placenta and small intestine but
absent from peripheral blood leukocytes, spleen and testis. Highest
expression was in the placental library. Comparison of the magic
roundabout sequence to that of roundabout revealed a transmembrane
protein with homology throughout but absence of some extracellular
domains. Thus, MR has two immunoglobulin and two fibronectin
domains in the extracellular domain compared to five immunoglobulin
and two fibronectin domains in the extracellular domains of the
neuronal specific roundabouts. A transmembrane domain was
identified by (i) using the transmembrane predicting software
PRED-TMR and (ii) using an alignment between human MR and human
ROBO1 peptide sequences. Both methods identified the same residues
as the transmembrane region of human MR as amino acids 468-490.
Thus, aa 1-467 are extracellular and aa 491-1007 are intracellular.
The intracellular domain contains a putative proline rich region
that is homologous to those in roundabout that are thought to
couple to c-ab1 (Bashaw et al (2000) Cell 101: 703-715).
[0594] Human SHGC-11739 (GenBank acc. G14646) sequence tagged site
(STS) was mapped to magic roundabout mRNA in a BLAST dbSTS search.
This STSmaps to chromosome 11 on the Stanford G3 physical map
(region 5647.00 cR10000 LOD 1.09 bin 129). Nevertheless, much
sequence is missing and the genomic structure is not known. Search
of the RIKEN database identified murine magic roundabout. The
predicted molecular weight for the peptide core of human MR was
107,457 kDa. This was confirmed by in vitro translation (FIG.
3).
EXAMPLE 5
ECSM4 Expression is Detectable in Tumours
[0595] In situ hybridisation was used to characterise expression of
ECSM4 in vivo. Expression of ECSM4 was found to be very restricted
(Table 13), with no signal detectable in many tissues including
neuronal tissue. In contrast, strong expression was detected in
pacenta and a range of tumours including those of the brain,
bladder and colonic metastasis to the liver (FIG. 27). Expression
within tumours was restricted to the tumour vasculature.
Immuno-histochemical staining of placenta confirmed endothelial
specific expression of the protein.
[0596] A search of CGAP SAGE libraries for ECSM4 detected it only
in endothelial and tumour libraries (Table 14). This was consistent
with in situ hybridisation results in the adult showing that
expression was restricted to tumour vessels (colon metastasis to
liver, ganglioglioma, bladder and breast carcinoma). TABLE-US-00015
TABLE 13 Expression of magic roundabout in human tissue in vivo.
Expression detected Placenta and umbilical cord foetal tissue (10.8
weeks menstrual age) Vessels in colorectal liver metastasis,
ganglioglioma, bladder and breast carcinoma. Expression not
detected Adult liver, brain cerebrum and large vessels, prostate,
colon, small bowel, heart, eye choroid and sclera, ovary, stomach,
breast
[0597] TABLE-US-00016 TABLE 14 CGAP SAGE libraries in which magic
roundabout was found on the basis of gene to tag mapping Library
Tags/million Tags HDMEC 171 HDMEC + VEGF 224 Medulloblastoma 102
Glioblastoma multiforme 85 Ovary, serous adenocarcinoma 59
Glioblastoma multiforme, pooled 48 HDMEC, human dermal
microvascular endothelial cells; VEGF, vascular endothelial growth
factor.
EXAMPLE 6
[0598] Induction of ECSM4 in hypoxic endothelial cells Initial
RT-PCR detected ECSM4 expression in endothelial but not other cell
lines such as fibroblasts (normal endometial and FEK4), colon
carcinoma (SW480 and HCT116), breast carcinoma (MDA453 and MDA468)
and HeLa cells. Ribonuclease protection analysis has confirmed and
extended this (FIG. 11a). ECSM4 expression was seen to be
restricted to endothelium (three different isolates) and absent
from fibroblast, carcinoma and neuronal cells. Induction of ECSM4
in hypoxia in endothelial (but not non-endothelial cells) was seen
when expression of ECSM4 was analysed using two different RNase
protection probes. Expression was on average 5.5 and 2.6 fold
higher in hypoxia for HUVEC and HDMEC respectively. Western
analysis identified a weak band of 110 kD in human dermal
microvascular endothelial cells (HDMEC) but absent from the
non-endothelial cells types (FIG. 11b). The band was more intense
when the HDMEC cells were epxosed to 18 h hyposia, consistent with
ECSM4 being a hypoxically regulated gene.
Sequence CWU 1
1
50 1 12 PRT Artificial Sequence Synthetically generated peptide 1
Gly Gly Asp Ser Leu Leu Gly Gly Arg Gly Ser Leu 1 5 10 2 20 PRT
Artificial Sequence Synthetically generated peptide 2 Leu Leu Gln
Pro Pro Ala Arg Gly His Ala His Asp Gly Gln Ala Leu 1 5 10 15 Ser
Thr Asp Leu 20 3 10 PRT Artificial Sequence Synthetically generated
peptide 3 Glu Pro Gln Asp Tyr Thr Glu Pro Val Glu 1 5 10 4 13 PRT
Artificial Sequence Synthetically generated peptide 4 Thr Ala Pro
Gly Gly Gln Gly Ala Pro Trp Ala Glu Glu 1 5 10 5 12 PRT Artificial
Sequence Synthetically generated peptide 5 Glu Arg Ala Thr Gln Glu
Pro Ser Glu His Gly Pro 1 5 10 6 17 PRT Artificial Sequence
Synthetically generated peptide 6 Leu Ser Gln Ser Pro Gly Ala Val
Pro Gln Ala Leu Val Ala Trp Arg 1 5 10 15 Ala 7 15 PRT Artificial
Sequence Synthetically generated peptide 7 Asp Ser Val Leu Thr Pro
Glu Glu Val Ala Leu Cys Leu Glu Leu 1 5 10 15 8 10 PRT Artificial
Sequence Synthetically generated peptide 8 Thr Tyr Gly Tyr Ile Ser
Val Pro Thr Ala 1 5 10 9 16 PRT Artificial Sequence Synthetically
generated peptide 9 Lys Gly Gly Val Leu Leu Cys Pro Pro Arg Pro Cys
Leu Thr Pro Thr 1 5 10 15 10 6 PRT Artificial Sequence
Synthetically generated peptide 10 Trp Leu Ala Asp Thr Trp 1 5 11
14 PRT Artificial Sequence Synthetically generated peptide 11 Trp
Leu Ala Asp Thr Trp Arg Ser Thr Ser Gly Ser Arg Asp 1 5 10 12 10
PRT Artificial Sequence Synthetically generated peptide 12 Ser Pro
Pro Thr Thr Tyr Gly Tyr Ile Ser 1 5 10 13 33 PRT Artificial
Sequence Synthetically generated peptide 13 Gly Ser Leu Ala Asn Gly
Trp Gly Ser Ala Ser Glu Asp Asn Ala Ala 1 5 10 15 Ser Ala Arg Ala
Ser Leu Val Ser Ser Ser Asp Gly Ser Phe Leu Ala 20 25 30 Asp 14 10
PRT Artificial Sequence Synthetically generated peptide 14 Phe Ala
Arg Ala Leu Ala Val Ala Val Asp 1 5 10 15 20 DNA Artificial
Sequence Primer 15 tgcagcttcc ttctcacctt 20 16 20 DNA Artificial
Sequence Primer 16 tcacatcccc attcacactg 20 17 22 DNA Artificial
Sequence Primer 17 tgtaccatga ggttctcaat gc 22 18 20 DNA Artificial
Sequence Primer 18 ttattgtggg ctcagaaggg 20 19 21 DNA Artificial
Sequence Primer 19 caaatgcttc caggtgaaaa a 21 20 20 DNA Artificial
Sequence Primer 20 cgttcaaagc atgaaatgga 20 21 1100 DNA Homo
sapiens CDS (152)...(460) 21 tgtctgctta tgcggtggct cgctgctcag
aacaggatgg cagagatgag caccaccatc 60 aaaaactcaa ggaccagtgc
tgtgggtcca gtcatctgtt tcatggaatt caccagtctg 120 gtatcttcaa
aatccagaag gatgatggca g atg gca gga agg agg aag agg 172 Met Ala Gly
Arg Arg Lys Arg 1 5 gta atc tgg aag agt ttc cgg acc tac tct gct gct
gtg att aaa caa 220 Val Ile Trp Lys Ser Phe Arg Thr Tyr Ser Ala Ala
Val Ile Lys Gln 10 15 20 cca cca gga aat ttt gat gac act gtt ctc
ctg agc tcc tcc ctt tcc 268 Pro Pro Gly Asn Phe Asp Asp Thr Val Leu
Leu Ser Ser Ser Leu Ser 25 30 35 tcg ggg aag aaa agc att gaa act
aca aaa ata aag tgt tat ttg gct 316 Ser Gly Lys Lys Ser Ile Glu Thr
Thr Lys Ile Lys Cys Tyr Leu Ala 40 45 50 55 gga gtg agg tct cat gtc
tgc tta tgc ggt ggc tcg ctg ctc aga aca 364 Gly Val Arg Ser His Val
Cys Leu Cys Gly Gly Ser Leu Leu Arg Thr 60 65 70 ggg aac cat tgg
aga tac tca tta ctc ttt gaa ggc tta cag tgg aat 412 Gly Asn His Trp
Arg Tyr Ser Leu Leu Phe Glu Gly Leu Gln Trp Asn 75 80 85 gaa ttc
aaa tac gac tta ttt gag gaa ttg aag ttg act tta tgg agc 460 Glu Phe
Lys Tyr Asp Leu Phe Glu Glu Leu Lys Leu Thr Leu Trp Ser 90 95 100
tgataagaat cttcttggag aaaaaaagac tggtacttct gaattaacca aaatcacagt
520 attctgaaga tgattctaca aagcctgctg tttctacaaa ggctgctgat
gatttctaca 580 aagcctgctg tagtgttgct gtggcctctg cttaaaaaag
tagaaaacac attgatgcag 640 catgttcacc ccaacctccc tgcctaaagg
cctcaggggc ccctccttgg gaagagggaa 700 gggcgccgtg aggattggta
aagagcccga attagggggg gatgggagtg gtgggagaat 760 aaggggacac
cttccatcct tgggatgctc accctgccca aattgacctt cctgatgaaa 820
ggccagctcc cagaaatgtg ccctacagtt acctactttc accctaaacc ctgcccttag
880 tcaaatcctt ttcttttttt aagcaatcaa cttcaattcc ttgtataacc
cccagtataa 940 aagggctttt ataccattct atcctattgc atgtaagcct
tgggtttggg aggtaacagt 1000 gtgggattcc cccatttcat ttccctgcca
cccaaacatg cctgtttttt tttaagcaat 1060 attaaatgtt tgtacttcag
aaaaaaaaaa aaaaaaaaaa 1100 22 103 PRT Homo sapiens 22 Met Ala Gly
Arg Arg Lys Arg Val Ile Trp Lys Ser Phe Arg Thr Tyr 1 5 10 15 Ser
Ala Ala Val Ile Lys Gln Pro Pro Gly Asn Phe Asp Asp Thr Val 20 25
30 Leu Leu Ser Ser Ser Leu Ser Ser Gly Lys Lys Ser Ile Glu Thr Thr
35 40 45 Lys Ile Lys Cys Tyr Leu Ala Gly Val Arg Ser His Val Cys
Leu Cys 50 55 60 Gly Gly Ser Leu Leu Arg Thr Gly Asn His Trp Arg
Tyr Ser Leu Leu 65 70 75 80 Phe Glu Gly Leu Gln Trp Asn Glu Phe Lys
Tyr Asp Leu Phe Glu Glu 85 90 95 Leu Lys Leu Thr Leu Trp Ser 100 23
1496 DNA Homo sapiens CDS (1)...(1395) 23 aac tgg ttg cga cac tgc
ggt gtt gca ctc tgg ctg ctg ctt ctg ggc 48 Asn Trp Leu Arg His Cys
Gly Val Ala Leu Trp Leu Leu Leu Leu Gly 1 5 10 15 acc gct gtg tgt
atc cac cgc cgt cgc cga gct agg gtg ctt ctg ggc 96 Thr Ala Val Cys
Ile His Arg Arg Arg Arg Ala Arg Val Leu Leu Gly 20 25 30 cca ggt
ctg tac aga tat acc agt gag gat gcc atc cta aaa cac agg 144 Pro Gly
Leu Tyr Arg Tyr Thr Ser Glu Asp Ala Ile Leu Lys His Arg 35 40 45
atg gat cac agt gac tcc cag tgg ttg gca gac act tgg cgt tcc acc 192
Met Asp His Ser Asp Ser Gln Trp Leu Ala Asp Thr Trp Arg Ser Thr 50
55 60 tct ggc tct cgg gac ctg agc agc agc agc agc ctc agc agt cgg
ctg 240 Ser Gly Ser Arg Asp Leu Ser Ser Ser Ser Ser Leu Ser Ser Arg
Leu 65 70 75 80 ggg gcg gat gcc cgg gac cca cta gac tgt cgt cgc tcc
ttg ctc tcc 288 Gly Ala Asp Ala Arg Asp Pro Leu Asp Cys Arg Arg Ser
Leu Leu Ser 85 90 95 tgg gac tcc cga agc ccc ggc gtg ccc ctg ctt
cca gac acc agc act 336 Trp Asp Ser Arg Ser Pro Gly Val Pro Leu Leu
Pro Asp Thr Ser Thr 100 105 110 ttt tat ggc tcc ctc atc gct gag ctg
ccc tcc agt acc cca gcc agg 384 Phe Tyr Gly Ser Leu Ile Ala Glu Leu
Pro Ser Ser Thr Pro Ala Arg 115 120 125 cca agt ccc cag gtc cca gct
gtc agg cgc ctc cca ccc cag ctg gcc 432 Pro Ser Pro Gln Val Pro Ala
Val Arg Arg Leu Pro Pro Gln Leu Ala 130 135 140 cag ctc tcc agc ccc
tgt tcc agc tca gac agc ctc tgc agc cgc agg 480 Gln Leu Ser Ser Pro
Cys Ser Ser Ser Asp Ser Leu Cys Ser Arg Arg 145 150 155 160 gga ctc
tct tct ccc cgc ttg tct ctg gcc cct gca gag gct tgg aag 528 Gly Leu
Ser Ser Pro Arg Leu Ser Leu Ala Pro Ala Glu Ala Trp Lys 165 170 175
gcc aaa aag aag cag gag ctg cag cat gcc aac agt tcc cca ctg ctc 576
Ala Lys Lys Lys Gln Glu Leu Gln His Ala Asn Ser Ser Pro Leu Leu 180
185 190 cgg ggc agc cac tcc ttg gag ctc cgg gcc tgt gag tta gga aat
aga 624 Arg Gly Ser His Ser Leu Glu Leu Arg Ala Cys Glu Leu Gly Asn
Arg 195 200 205 ggt tcc aag aac ctt tcc caa agc cca ggg gct gtg ccc
caa gct ctg 672 Gly Ser Lys Asn Leu Ser Gln Ser Pro Gly Ala Val Pro
Gln Ala Leu 210 215 220 gtt gcc tgg cgg gcc ctg gga ccg aaa ctc ctc
agc tcc tca aat gag 720 Val Ala Trp Arg Ala Leu Gly Pro Lys Leu Leu
Ser Ser Ser Asn Glu 225 230 235 240 ctg gtt act cgt cat ctc cct cca
gca ccc ctc ttt cct cat gaa act 768 Leu Val Thr Arg His Leu Pro Pro
Ala Pro Leu Phe Pro His Glu Thr 245 250 255 ccc cca act cag agt caa
cag acc cag cct ccg gtg gca cca cag gct 816 Pro Pro Thr Gln Ser Gln
Gln Thr Gln Pro Pro Val Ala Pro Gln Ala 260 265 270 ccc tcc tcc atc
ctg ctg cca gca gcc ccc atc ccc atc ctt agc ccc 864 Pro Ser Ser Ile
Leu Leu Pro Ala Ala Pro Ile Pro Ile Leu Ser Pro 275 280 285 tgc agt
ccc cct agc ccc cag gcc tct tcc ctc tct ggc ccc agc cca 912 Cys Ser
Pro Pro Ser Pro Gln Ala Ser Ser Leu Ser Gly Pro Ser Pro 290 295 300
gct tcc agt cgc ctg tcc agc tcc tca ctg tca tcc ctg ggg gag gat 960
Ala Ser Ser Arg Leu Ser Ser Ser Ser Leu Ser Ser Leu Gly Glu Asp 305
310 315 320 caa gac agc gtg ctg acc cct gag gag gta gcc ctg tgc ttg
gaa ctc 1008 Gln Asp Ser Val Leu Thr Pro Glu Glu Val Ala Leu Cys
Leu Glu Leu 325 330 335 agt gag ggt gag gag act ccc agg aac agc gtc
tct ccc atg cca agg 1056 Ser Glu Gly Glu Glu Thr Pro Arg Asn Ser
Val Ser Pro Met Pro Arg 340 345 350 gct cct tca ccc ccc acc acc tat
ggg tac atc agc gtc cca aca gcc 1104 Ala Pro Ser Pro Pro Thr Thr
Tyr Gly Tyr Ile Ser Val Pro Thr Ala 355 360 365 tca gag ttc acg gac
atg ggc agg act gga gga ggg gtg ggg ccc aag 1152 Ser Glu Phe Thr
Asp Met Gly Arg Thr Gly Gly Gly Val Gly Pro Lys 370 375 380 ggg gga
gtc ttg ctg tgc cca cct cgg ccc tgc ctc acc ccc acc ccc 1200 Gly
Gly Val Leu Leu Cys Pro Pro Arg Pro Cys Leu Thr Pro Thr Pro 385 390
395 400 agc gag ggc tcc tta gcc aat ggt tgg ggc tca gcc tct gag gac
aat 1248 Ser Glu Gly Ser Leu Ala Asn Gly Trp Gly Ser Ala Ser Glu
Asp Asn 405 410 415 gcc gcc agc gcc aga gcc agc ctt gtc agc tcc tcc
gat ggc tcc ttc 1296 Ala Ala Ser Ala Arg Ala Ser Leu Val Ser Ser
Ser Asp Gly Ser Phe 420 425 430 ctc gct gat gct cac ttt gcc cgg gcc
ctg gca gtg gct gtg gat agc 1344 Leu Ala Asp Ala His Phe Ala Arg
Ala Leu Ala Val Ala Val Asp Ser 435 440 445 ttt ggt ttc ggt cta gag
ccc agg gag gca gac tgc gtc ttc ata ggt 1392 Phe Gly Phe Gly Leu
Glu Pro Arg Glu Ala Asp Cys Val Phe Ile Gly 450 455 460 atg
tgaggtctcc ccatcttact cctcactcat gccccttgcc tttctaacaa 1445 Met 465
ctgttatcat gtcatcattg ttaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1496 24
465 PRT Homo sapiens 24 Asn Trp Leu Arg His Cys Gly Val Ala Leu Trp
Leu Leu Leu Leu Gly 1 5 10 15 Thr Ala Val Cys Ile His Arg Arg Arg
Arg Ala Arg Val Leu Leu Gly 20 25 30 Pro Gly Leu Tyr Arg Tyr Thr
Ser Glu Asp Ala Ile Leu Lys His Arg 35 40 45 Met Asp His Ser Asp
Ser Gln Trp Leu Ala Asp Thr Trp Arg Ser Thr 50 55 60 Ser Gly Ser
Arg Asp Leu Ser Ser Ser Ser Ser Leu Ser Ser Arg Leu 65 70 75 80 Gly
Ala Asp Ala Arg Asp Pro Leu Asp Cys Arg Arg Ser Leu Leu Ser 85 90
95 Trp Asp Ser Arg Ser Pro Gly Val Pro Leu Leu Pro Asp Thr Ser Thr
100 105 110 Phe Tyr Gly Ser Leu Ile Ala Glu Leu Pro Ser Ser Thr Pro
Ala Arg 115 120 125 Pro Ser Pro Gln Val Pro Ala Val Arg Arg Leu Pro
Pro Gln Leu Ala 130 135 140 Gln Leu Ser Ser Pro Cys Ser Ser Ser Asp
Ser Leu Cys Ser Arg Arg 145 150 155 160 Gly Leu Ser Ser Pro Arg Leu
Ser Leu Ala Pro Ala Glu Ala Trp Lys 165 170 175 Ala Lys Lys Lys Gln
Glu Leu Gln His Ala Asn Ser Ser Pro Leu Leu 180 185 190 Arg Gly Ser
His Ser Leu Glu Leu Arg Ala Cys Glu Leu Gly Asn Arg 195 200 205 Gly
Ser Lys Asn Leu Ser Gln Ser Pro Gly Ala Val Pro Gln Ala Leu 210 215
220 Val Ala Trp Arg Ala Leu Gly Pro Lys Leu Leu Ser Ser Ser Asn Glu
225 230 235 240 Leu Val Thr Arg His Leu Pro Pro Ala Pro Leu Phe Pro
His Glu Thr 245 250 255 Pro Pro Thr Gln Ser Gln Gln Thr Gln Pro Pro
Val Ala Pro Gln Ala 260 265 270 Pro Ser Ser Ile Leu Leu Pro Ala Ala
Pro Ile Pro Ile Leu Ser Pro 275 280 285 Cys Ser Pro Pro Ser Pro Gln
Ala Ser Ser Leu Ser Gly Pro Ser Pro 290 295 300 Ala Ser Ser Arg Leu
Ser Ser Ser Ser Leu Ser Ser Leu Gly Glu Asp 305 310 315 320 Gln Asp
Ser Val Leu Thr Pro Glu Glu Val Ala Leu Cys Leu Glu Leu 325 330 335
Ser Glu Gly Glu Glu Thr Pro Arg Asn Ser Val Ser Pro Met Pro Arg 340
345 350 Ala Pro Ser Pro Pro Thr Thr Tyr Gly Tyr Ile Ser Val Pro Thr
Ala 355 360 365 Ser Glu Phe Thr Asp Met Gly Arg Thr Gly Gly Gly Val
Gly Pro Lys 370 375 380 Gly Gly Val Leu Leu Cys Pro Pro Arg Pro Cys
Leu Thr Pro Thr Pro 385 390 395 400 Ser Glu Gly Ser Leu Ala Asn Gly
Trp Gly Ser Ala Ser Glu Asp Asn 405 410 415 Ala Ala Ser Ala Arg Ala
Ser Leu Val Ser Ser Ser Asp Gly Ser Phe 420 425 430 Leu Ala Asp Ala
His Phe Ala Arg Ala Leu Ala Val Ala Val Asp Ser 435 440 445 Phe Gly
Phe Gly Leu Glu Pro Arg Glu Ala Asp Cys Val Phe Ile Gly 450 455 460
Met 465 25 2076 DNA Homo sapiens 25 aggggactct cttctccccg
cttgtctctg gcccctgcag aggcttggaa ggccaaaaag 60 aaagcaggag
ctgcagcatg ccaacagttc cccactgctc cggggcagcc actccttaga 120
gctccgggcc tgtgagttag gaaatagagg ttccaagaac ctttcccaaa gcccaggagc
180 tgtgccccaa gctctggttg cctggcgggc cctgggaccg aaactcctca
gctcctcaaa 240 tgagctggtt actcgtcatc tccctccagc acccctcttt
cctcatgaaa ctcccccaac 300 tcagagtcaa cagacccagc ctccggtggc
accacaggct ccctcctcca tcctgctgcc 360 agcagccccc atccccatcc
ttagcccctg cagtccccct agcccccagg cctcttccct 420 ctctggcccc
agcccagctt ccagtcgcct gtccagctcc tcactgtcat ccctggggga 480
ggatcaagac agcgtgctga cccctgagga ggtagccctg tgcttggaac tcagtgaggg
540 tgaggagact cccaggaaca gcgtctctcc catgccaagg gttccttcac
cccccaccac 600 ctatgggtac atcagcgtcc caacagcctc agagttcacg
gacatgggca ggactggagg 660 aggggtgggg cccaaggggg gagtcttgct
gtgcccacct cggccctgcc tcacccccac 720 ccccagcgag ggctccttag
ccaatggttg gggctcagcc tctgaggaca atgccgccag 780 cgccagagcc
agccttgtca gctcctccga tggctccttc ctcgctgatg ctcactttgc 840
ccgggccctg gcagtggctg tggatagctt tggtttcggt ctagagccca gggaggcaga
900 ctgcgtcttc atagatgcct catcacctcc ctccccacgg gattgagatc
ttcctgaccc 960 ccaacctctc cctgcccctg tgggaagtgg aggccagact
ggttggaaga caatggaagg 1020 tcagccacac ccagcggctg ggaaggggga
tgcctccctg gccccctgac tctcagatct 1080 cttcccagag aagtcagctc
cactgtcgta tgcccaaggg tgggtgcttc tcctgtagat 1140 tactcctgaa
ccgtgtccct gagacttccc agacgggaat cagaaccact tctcctgtcc 1200
acccacaaga cctgggctgt ggtgtgtggg tcttggcctg tgtttctctg cagctggggt
1260 ccaccttccc aagcctccag agagttctcc ctccacgatt gtgaaaacaa
atgaaaacaa 1320 aattagagca aagctgtacc tgggagccct cagggagcaa
aacatcatct ccacctgact 1380 cctagccact gctttctcct ctgtgccatc
cactcccacc acccaggttg tttttggcct 1440 gaaggagcaa gccctgcctg
ctggcttttc cccccaacca tttgggattc acagggaagt 1500 gggagggagc
ccagagggtg gccttttgtg ggagggacag cagtggctgc tgggggagag 1560
ggctgtggag gaaggagctt ctcggagccc cctctcagcc ttacctgggc ccctcctcta
1620 gagaagagct caactctctc ccaaccctca ccaatggaaa gaaaataatt
atgaatgccg 1680 actgaggcac tgaggcccct acctcatgcc caaaacaaag
gggttcaagg ctgggtctag 1740 cgaggatgct tgaaggaagg gaggtatgga
gcccgtaggt caaaagcacc catcctcgta 1800 ctgttgtcac tatgagctta
agaaatttga taccataaaa tggtaaagac ttgagttctg 1860 tgagatcatt
ccccggagca ccatttttag gggagcacct ggagagatgg caagaatttc 1920
ctgagttagg cagggatcag gcattcattg acactcaggg agtgtcacac atttctgttc
1980 tgcaattaaa gggagaatga ggttcatcca ccaaatttta agcagaatat
aggaagggca 2040 ggggtgggga gtttcagggt ctgctggtcc tgggca 2076 26 314
PRT Homo sapiens 26 Gly Asp Ser Leu Leu Pro Ala Cys Leu Trp Pro Leu
Gln Arg Leu Gly 1 5 10 15 Arg Pro Lys Arg Lys Gln Glu Leu Gln
His
Ala Asn Ser Ser Pro Leu 20 25 30 Leu Arg Gly Ser His Ser Leu Glu
Leu Arg Ala Cys Glu Leu Gly Asn 35 40 45 Arg Gly Ser Lys Asn Leu
Ser Gln Ser Pro Gly Ala Val Pro Gln Ala 50 55 60 Leu Val Ala Trp
Arg Ala Leu Gly Pro Lys Leu Leu Ser Ser Ser Asn 65 70 75 80 Glu Leu
Val Thr Arg His Leu Pro Pro Ala Pro Leu Phe Pro His Glu 85 90 95
Thr Pro Pro Thr Gln Ser Gln Gln Thr Gln Pro Pro Val Ala Pro Gln 100
105 110 Ala Pro Ser Ser Ile Leu Leu Pro Ala Ala Pro Ile Pro Ile Leu
Ser 115 120 125 Pro Cys Ser Pro Pro Ser Pro Gln Ala Ser Ser Leu Ser
Gly Pro Ser 130 135 140 Pro Ala Ser Ser Arg Leu Ser Ser Ser Ser Leu
Ser Ser Leu Gly Glu 145 150 155 160 Asp Gln Asp Ser Val Leu Thr Pro
Glu Glu Val Ala Leu Cys Leu Glu 165 170 175 Leu Ser Glu Gly Glu Glu
Thr Pro Arg Asn Ser Val Ser Pro Met Pro 180 185 190 Arg Val Pro Ser
Pro Pro Thr Thr Tyr Gly Tyr Ile Ser Val Pro Thr 195 200 205 Ala Ser
Glu Phe Thr Asp Met Gly Arg Thr Gly Gly Gly Val Gly Pro 210 215 220
Lys Gly Gly Val Leu Leu Cys Pro Pro Arg Pro Cys Leu Thr Pro Thr 225
230 235 240 Pro Ser Glu Gly Ser Leu Ala Asn Gly Trp Gly Ser Ala Ser
Glu Asp 245 250 255 Asn Ala Ala Ser Ala Arg Ala Ser Leu Val Ser Ser
Ser Asp Gly Ser 260 265 270 Phe Leu Ala Asp Ala His Phe Ala Arg Ala
Leu Ala Val Ala Val Asp 275 280 285 Ser Phe Gly Phe Gly Leu Glu Pro
Arg Glu Ala Asp Cys Val Phe Ile 290 295 300 Asp Ala Ser Ser Pro Pro
Ser Pro Arg Asp 305 310 27 1046 DNA Homo sapiens 27 tccagctcag
acagcctctg cagccgcagg ggactctctt ctccccgctt gtctctggcc 60
cctgcagagg cttggaaggc caaaaagaag caggagctgc agcatgccaa cagttcccca
120 ctgctccggg gcagccactc cttggagctc cgggcctgtg agttaggaaa
tagaggttcc 180 aagaaccttt cccaaagccc aggggctgtg ccccaagctc
tggttgcctg gcgggccctg 240 ggaccgaaac tcctcagctc ctcaaatgag
ctggttactc gtcatctccc tccagcaccc 300 ctctttcctc atgaaactcc
cccaactcag agtcaacaga cccagcctcc ggtggcacca 360 caggctccct
cctccatcct gctgccagca gcccccatcc ccatccttag cccctgcagt 420
ccccctagcc cccaggcctc ttccctctct ggccccagcc cagcttccag tcgcctgtcc
480 agctcctcac tgtcatccct gggggaggat caagacagcg tgctgacccc
tgaggaggta 540 gccctgtgct tggaactcag tgagggtgag gagactccca
ggaacagcgt ctctcccatg 600 ccaagggctc cttcaccccc caccacctat
gggtacatca gcgtcccaac agcctcagag 660 ttcacggaca tgggcaggac
tggaggaggg gtggggccca aggggggagt cttgctgtgc 720 ccacctcggc
cctgcctcac ccccaccccc agcgagggct ccttagccaa tggttggggc 780
tcagcctctg aggacaatgc cgccagcgcc agagccagcc ttgtcagctc ctccgatggc
840 tccttcctcg ctgatgctca ctttgcccgg gccctggcag tggctgtgga
tagctttggt 900 ttcggtctag agcccaggga ggcagactgc gtcttcatag
gtatgtgagg tctccccatc 960 ttactcctca ctcatgcccc ttgcctttct
aacaactgtt atcatgtcat cattgttaaa 1020 aaaaaaaaaa aaaaaaaaaa aaaaaa
1046 28 1023 DNA Homo sapiens 28 aggggactct cttctccccg cttgtctctg
gcccctgcag aggcttggaa ggccaaaaag 60 aaagcaggag ctgcagcatg
ccaacagttc cccactgctc cggggcagcc actccttaga 120 gctccgggcc
tgtgagttag gaaatagagg ttccaagaac ctttcccaaa gcccaggagc 180
tgtgccccaa gctctggttg cctggcgggc cctgggaccg aaactcctca gctcctcaaa
240 tgagctggtt actcgtcatc tccctccagc acccctcttt cctcatgaaa
ctcccccaac 300 tcagagtcaa cagacccagc ctccggtggc accacaggct
ccctcctcca tcctgctgcc 360 agcagccccc atccccatcc ttagcccctg
cagtccccct agcccccagg cctcttccct 420 ctctggcccc agcccagctt
ccagtcgcct gtccagctcc tcactgtcat ccctggggga 480 ggatcaagac
agcgtgctga cccctgagga ggtagccctg tgcttggaac tcagtgaggg 540
tgaggagact cccaggaaca gcgtctctcc catgccaagg gttccttcac cccccaccac
600 ctatgggtac atcagcgtcc caacagcctc agagttcacg gacatgggca
ggactggagg 660 aggggtgggg cccaaggggg gagtcttgct gtgcccacct
cggccctgcc tcacccccac 720 ccccagcgag ggctccttag ccaatggttg
gggctcagcc tctgaggaca atgccgccag 780 cgccagagcc agccttgtca
gctcctccga tggctccttc ctcgctgatg ctcactttgc 840 ccgggccctg
gcagtggctg tggatagctt tggtttcggt ctagagccca gggaggcaga 900
ctgcgtcttc atagatgcct catcacctcc ctccccacgg gattgagatc ttcctgaccc
960 ccaacctctc cctgcccctg tgggaagtgg aggccagact ggttggaaga
caatggaagg 1020 tca 1023 29 1271 DNA Mus musculus 29 gggtctttac
agttttatag aattaagttc cttaagctca gagtgggggt agaaatgaga 60
atagggaatt ggttccctgt cttcctgcgt ccttatcctt tcagtctcct ccaatgattt
120 cactttgaag gattgaatgt gaggctgtat aggggccagt gcatccagaa
cgtttctcca 180 taagtttcct tggatggttg tgaatgggga aagggttgag
ttggtgttgt aagggaggag 240 tccaagttaa tattagaggg gtcttccaca
ggtccaccaa cagaggccct caccaaaaaa 300 catttctgtc cttcctgaag
acctggttgg cttcccttct ttccatgatc cacttaggcg 360 ggagctccgg
agccaggctt acttaggcca aaggttctgg ttgtggagag tctgctgtcc 420
tgaagatgct gtcttgttct cagtgggaat ccaagactcc cgtgatcata ttttggtttg
480 ctttcattta ttttaacaat cccaatgaca gagctctcca gaagcctagt
gacagtggac 540 ttctattaca gagaagcata ggccaagacc tccacatgtg
agaaagccag gggacagaca 600 ggagagtggt ctgggtgctc ttctggcctt
ctcagggaca attcaggagg aatcacacag 660 ccttgggcac agcaccagtt
agccaacttc gctgggaaga ggccctagaa tcaggaggcc 720 agggaggcag
ccccctcccc agcctctggg tgtggctgat ctcagcatct tccaaccagt 780
ctggcctcca ctcccacaaa ggcagagaga agcttcgggt cagggagaga tcaccccgag
840 gggagggagg tgatgaggca tcagtgaaga cacagtcagc ttccctggga
tccagactga 900 ggccaaagct atccacagcc actgccaggg cacgagcaaa
gtgagtatca gcgaggaagg 960 agccatcaga agagctaacc aggctggccc
tggcgctggg gacattgtcc tcagaagctg 1020 agccccaacc attggccagg
gagccctcgc tgggtgtagg ggtggggcag ggccgaggtg 1080 gatacagtaa
gttcccaacc tcagacccca cgcccccgcc agctctgccc atgtctgcca 1140
gtcctgagca ggttggtatg ctgatatagc cataggttgt tggcggggaa ggagctcttg
1200 gcataggaga tacactgttc gtgggtgtct cctccccatc actgagctcc
agacacaggg 1260 ctacctcctc g 1271 30 205 PRT Mus musculus 30 Glu
Glu Val Ala Leu Cys Leu Glu Leu Ser Asp Gly Glu Glu Thr Pro 1 5 10
15 Thr Asn Ser Val Ser Pro Met Pro Arg Ala Pro Ser Pro Pro Thr Thr
20 25 30 Tyr Gly Tyr Ile Ser Ile Pro Thr Cys Ser Gly Leu Ala Asp
Met Gly 35 40 45 Arg Ala Gly Gly Gly Val Gly Ser Glu Val Gly Asn
Leu Leu Tyr Pro 50 55 60 Pro Arg Pro Cys Pro Thr Pro Thr Pro Ser
Glu Gly Ser Leu Ala Asn 65 70 75 80 Gly Trp Gly Ser Ala Ser Glu Asp
Asn Val Pro Ser Ala Arg Ala Ser 85 90 95 Leu Val Ser Ser Ser Asp
Gly Ser Phe Leu Ala Asp Thr His Phe Ala 100 105 110 Arg Ala Leu Ala
Val Ala Val Asp Ser Phe Gly Leu Ser Leu Asp Pro 115 120 125 Arg Glu
Ala Asp Cys Val Phe Thr Asp Ala Ser Ser Pro Pro Ser Pro 130 135 140
Arg Gly Asp Leu Ser Leu Thr Arg Ser Phe Ser Leu Pro Leu Trp Glu 145
150 155 160 Trp Arg Pro Asp Trp Leu Glu Asp Ala Glu Ile Ser His Thr
Gln Arg 165 170 175 Leu Gly Arg Gly Leu Pro Pro Trp Pro Pro Asp Ser
Arg Ala Ser Ser 180 185 190 Gln Arg Ser Trp Leu Thr Gly Ala Val Pro
Lys Ala Val 195 200 205 31 417 PRT Homo sapiens 31 Met Asp His Ser
Asp Ser Gln Trp Leu Ala Asp Thr Trp Arg Ser Thr 1 5 10 15 Ser Gly
Ser Arg Asp Leu Ser Ser Ser Ser Ser Leu Ser Ser Arg Leu 20 25 30
Gly Ala Asp Ala Arg Asp Pro Leu Asp Cys Arg Arg Ser Leu Leu Ser 35
40 45 Trp Asp Ser Arg Ser Pro Gly Val Pro Leu Leu Pro Asp Thr Ser
Thr 50 55 60 Phe Tyr Gly Ser Leu Ile Ala Glu Leu Pro Ser Ser Thr
Pro Ala Arg 65 70 75 80 Pro Ser Pro Gln Val Pro Ala Val Arg Arg Leu
Pro Pro Gln Leu Ala 85 90 95 Gln Leu Ser Ser Pro Cys Ser Ser Ser
Asp Ser Leu Cys Ser Arg Arg 100 105 110 Gly Leu Ser Ser Pro Arg Leu
Ser Leu Ala Pro Ala Glu Ala Trp Lys 115 120 125 Ala Lys Lys Lys Gln
Glu Leu Gln His Ala Asn Ser Ser Pro Leu Leu 130 135 140 Arg Gly Ser
His Ser Leu Glu Leu Arg Ala Cys Glu Leu Gly Asn Arg 145 150 155 160
Gly Ser Lys Asn Leu Ser Gln Ser Pro Gly Ala Val Pro Gln Ala Leu 165
170 175 Val Ala Trp Arg Ala Leu Gly Pro Lys Leu Leu Ser Ser Ser Asn
Glu 180 185 190 Leu Val Thr Arg His Leu Pro Pro Ala Pro Leu Phe Pro
His Glu Thr 195 200 205 Pro Pro Thr Gln Ser Gln Gln Thr Gln Pro Pro
Val Ala Pro Gln Ala 210 215 220 Pro Ser Ser Ile Leu Leu Pro Ala Ala
Pro Ile Pro Ile Leu Ser Pro 225 230 235 240 Cys Ser Pro Pro Ser Pro
Gln Ala Ser Ser Leu Ser Gly Pro Ser Pro 245 250 255 Ala Ser Ser Arg
Leu Ser Ser Ser Ser Leu Ser Ser Leu Gly Glu Asp 260 265 270 Gln Asp
Ser Val Leu Thr Pro Glu Glu Val Ala Leu Cys Leu Glu Leu 275 280 285
Ser Glu Gly Glu Glu Thr Pro Arg Asn Ser Val Ser Pro Met Pro Arg 290
295 300 Ala Pro Ser Pro Pro Thr Thr Tyr Gly Tyr Ile Ser Val Pro Thr
Ala 305 310 315 320 Ser Glu Phe Thr Asp Met Gly Arg Thr Gly Gly Gly
Val Gly Pro Lys 325 330 335 Gly Gly Val Leu Leu Cys Pro Pro Arg Pro
Cys Leu Thr Pro Thr Pro 340 345 350 Ser Glu Gly Ser Leu Ala Asn Gly
Trp Gly Ser Ala Ser Glu Asp Asn 355 360 365 Ala Ala Ser Ala Arg Ala
Ser Leu Val Ser Ser Ser Asp Gly Ser Phe 370 375 380 Leu Ala Asp Ala
His Phe Ala Arg Ala Leu Ala Val Ala Val Asp Ser 385 390 395 400 Phe
Gly Phe Gly Leu Glu Pro Arg Glu Ala Asp Cys Val Phe Ile Gly 405 410
415 Met 32 516 PRT Mus musculus 32 Pro Thr Val Thr Tyr Gln Arg Gly
Gly Glu Ala Val Ser Ser Gly Gly 1 5 10 15 Arg Pro Gly Leu Leu Asn
Ile Ser Glu Pro Ala Thr Gln Pro Trp Leu 20 25 30 Ala Asp Thr Trp
Pro Asn Thr Gly Asn Asn His Asn Asp Cys Ser Ile 35 40 45 Asn Cys
Cys Thr Ala Gly Asn Gly Asn Ser Asp Ser Asn Leu Thr Thr 50 55 60
Tyr Ser Arg Pro Ala Asp Cys Ile Ala Asn Tyr Asn Asn Gln Leu Asp 65
70 75 80 Asn Lys Gln Thr Asn Leu Met Leu Pro Glu Ser Thr Val Tyr
Gly Asp 85 90 95 Val Asp Leu Ser Asn Lys Ile Asn Glu Met Lys Thr
Phe Asn Ser Pro 100 105 110 Asn Leu Lys Asp Gly Arg Phe Val Asn Pro
Ser Gly Gln Pro Thr Pro 115 120 125 Tyr Ala Thr Thr Gln Leu Ile Gln
Ala Asn Leu Ser Asn Asn Met Asn 130 135 140 Asn Gly Ala Gly Asp Ser
Ser Glu Lys His Trp Lys Pro Pro Gly Gln 145 150 155 160 Gln Lys Pro
Glu Val Ala Pro Ile Gln Tyr Asn Ile Met Glu Gln Asn 165 170 175 Lys
Leu Asn Lys Asp Tyr Arg Ala Asn Asp Thr Ile Pro Pro Thr Ile 180 185
190 Pro Tyr Asn Gln Ser Tyr Asp Gln Asn Thr Gly Gly Ser Tyr Asn Ser
195 200 205 Ser Asp Arg Gly Ser Ser Thr Ser Gly Ser Gln Gly His Lys
Lys Gly 210 215 220 Ala Arg Thr Pro Lys Ala Pro Lys Gln Gly Gly Met
Asn Trp Ala Asp 225 230 235 240 Leu Leu Pro Pro Pro Pro Ala His Pro
Pro Pro His Ser Asn Ser Glu 245 250 255 Glu Tyr Asn Met Ser Val Asp
Glu Ser Tyr Asp Gln Glu Met Pro Cys 260 265 270 Pro Val Pro Pro Ala
Pro Met Tyr Leu Gln Gln Asp Glu Leu Gln Glu 275 280 285 Glu Glu Asp
Glu Arg Gly Pro Thr Pro Pro Val Arg Gly Ala Ala Ser 290 295 300 Ser
Pro Ala Ala Val Ser Tyr Ser His Gln Ser Thr Ala Thr Leu Thr 305 310
315 320 Pro Ser Pro Gln Glu Glu Leu Gln Pro Met Leu Gln Asp Cys Pro
Glu 325 330 335 Asp Leu Gly His Met Pro His Pro Pro Asp Arg Arg Arg
Gln Pro Val 340 345 350 Ser Pro Pro Pro Pro Pro Arg Pro Ile Ser Pro
Pro His Thr Tyr Gly 355 360 365 Tyr Ile Ser Gly Pro Leu Val Ser Asp
Met Asp Thr Asp Ala Pro Glu 370 375 380 Glu Glu Glu Asp Glu Ala Asp
Met Glu Val Ala Lys Met Gln Thr Arg 385 390 395 400 Arg Leu Leu Leu
Arg Gly Leu Glu Gln Thr Pro Ala Ser Ser Val Gly 405 410 415 Asp Leu
Glu Ser Ser Val Thr Gly Ser Met Ile Asn Gly Trp Gly Ser 420 425 430
Ala Ser Glu Glu Asp Asn Ile Ser Ser Gly Arg Ser Ser Val Ser Ser 435
440 445 Ser Asp Gly Ser Phe Phe Thr Asp Ala Asp Phe Ala Gln Ala Val
Ala 450 455 460 Ala Ala Ala Glu Tyr Ala Gly Leu Lys Val Ala Arg Arg
Gln Met Gln 465 470 475 480 Asp Ala Ala Gly Arg Arg His Phe His Ala
Ser Gln Cys Pro Arg Pro 485 490 495 Thr Ser Pro Val Ser Thr Asp Ser
Asn Met Ser Ala Val Val Ile Gln 500 505 510 Lys Ala Arg Pro 515 33
297 PRT Mus musculus 33 Thr Ala Thr Leu Thr Pro Ser Pro Gln Glu Glu
Leu Gln Pro Met Leu 1 5 10 15 Gln Asp Cys Pro Glu Asp Leu Gly His
Met Pro His Pro Pro Asp Arg 20 25 30 Arg Arg Gln Pro Val Ser Pro
Pro Pro Pro Pro Arg Pro Ile Ser Pro 35 40 45 Pro His Thr Tyr Gly
Tyr Ile Ser Gly Pro Leu Val Ser Asp Met Asp 50 55 60 Thr Asp Ala
Pro Glu Glu Glu Glu Asp Glu Ala Asp Met Glu Val Ala 65 70 75 80 Lys
Met Gln Thr Arg Arg Leu Leu Leu Arg Gly Leu Glu Gln Thr Pro 85 90
95 Ala Ser Ser Val Gly Asp Leu Glu Ser Ser Val Thr Gly Ser Met Ile
100 105 110 Asn Gly Trp Gly Ser Ala Ser Glu Glu Asp Asn Ile Ser Ser
Gly Arg 115 120 125 Ser Ser Val Ser Ser Ser Asp Gly Ser Phe Phe Thr
Asp Ala Asp Phe 130 135 140 Ala Gln Ala Val Ala Ala Ala Ala Glu Tyr
Ala Gly Leu Lys Val Ala 145 150 155 160 Arg Arg Gln Met Gln Asp Ala
Ala Gly Arg Arg His Phe His Ala Ser 165 170 175 Gln Cys Pro Arg Pro
Thr Ser Pro Val Ser Thr Asp Ser Asn Met Ser 180 185 190 Ala Val Val
Ile Gln Lys Ala Arg Pro Ala Lys Lys Gln Lys His Gln 195 200 205 Pro
Gly His Leu Arg Arg Glu Ala Tyr Ala Asp Asp Leu Pro Pro Pro 210 215
220 Pro Val Pro Pro Pro Ala Ile Lys Ser Pro Thr Val Gln Ser Lys Ala
225 230 235 240 Gln Leu Glu Val Arg Pro Val Met Val Pro Lys Leu Ala
Ser Ile Glu 245 250 255 Ala Arg Thr Asp Arg Ser Ser Asp Arg Lys Gly
Gly Ser Tyr Lys Gly 260 265 270 Arg Glu Ala Leu Asp Gly Arg Gln Val
Thr Asp Leu Arg Thr Asn Pro 275 280 285 Ser Asp Pro Arg Glu Ala Gln
Glu Gln 290 295 34 135 PRT Mus musculus 34 Glu Glu Val Ala Leu Cys
Leu Glu Leu Ser Asp Gly Glu Glu Thr Pro 1 5 10 15 Thr Asn Ser Val
Ser Pro Met Pro Arg Ala Pro Ser Pro Pro Thr Thr 20 25 30 Tyr Gly
Tyr Ile Ser Ile Pro Thr Cys Ser Gly Leu Ala Asp Met Gly 35 40 45
Arg Ala Gly Gly Gly Val Gly Ser Glu Val Gly Asn Leu Leu Tyr Pro 50
55 60 Pro Arg Pro Cys Pro Thr Pro Thr Pro Ser Glu Gly Ser Leu Ala
Asn 65 70 75 80 Gly Trp Gly Ser Ala Ser Glu Asp Asn Val Pro Ser Ala
Arg Ala Ser 85 90 95 Leu Val Ser Ser Ser Asp Gly Ser Phe Leu Ala
Asp Thr His Phe Ala 100 105 110 Arg Ala Leu Ala Val Ala Val Asp Ser
Phe Gly Leu Ser Leu Asp Pro 115 120 125 Arg Glu Ala
Asp Cys Val Phe 130 135 35 135 PRT Homo sapiens 35 Glu Glu Val Ala
Leu Cys Leu Glu Leu Ser Glu Gly Glu Glu Thr Pro 1 5 10 15 Arg Asn
Ser Val Ser Pro Met Pro Arg Ala Pro Ser Pro Pro Thr Thr 20 25 30
Tyr Gly Tyr Ile Ser Val Pro Thr Ala Ser Glu Phe Thr Asp Met Gly 35
40 45 Arg Thr Gly Gly Gly Val Gly Pro Lys Gly Gly Val Leu Leu Cys
Pro 50 55 60 Pro Arg Pro Cys Leu Thr Pro Thr Pro Ser Glu Gly Ser
Leu Ala Asn 65 70 75 80 Gly Trp Gly Ser Ala Ser Glu Asp Asn Ala Ala
Ser Ala Arg Ala Ser 85 90 95 Leu Val Ser Ser Ser Asp Gly Ser Phe
Leu Ala Asp Ala His Phe Ala 100 105 110 Arg Ala Leu Ala Val Ala Val
Asp Ser Phe Gly Phe Gly Leu Glu Pro 115 120 125 Arg Glu Ala Asp Cys
Val Phe 130 135 36 3715 DNA Homo sapiens CDS (70)...(3381) 36
gcggccgcga attcggcacg agcagcagga caaagtgctc gggacaagga catagggctg
60 agagtagcc atg ggc tct gga gga gac agc ctc ctg ggg ggc agg ggt
tcc 111 Met Gly Ser Gly Gly Asp Ser Leu Leu Gly Gly Arg Gly Ser 1 5
10 ctg cct ctg ctg ctc ctg ctc atc atg gga ggc atg gct cag gac tcc
159 Leu Pro Leu Leu Leu Leu Leu Ile Met Gly Gly Met Ala Gln Asp Ser
15 20 25 30 ccg ccc cag atc cta gtc cac ccc cag gac cag ctg ttc cag
ggc cct 207 Pro Pro Gln Ile Leu Val His Pro Gln Asp Gln Leu Phe Gln
Gly Pro 35 40 45 ggc cct gcc agg atg agc tgc caa gcc tca ggc cag
cca cct ccc acc 255 Gly Pro Ala Arg Met Ser Cys Gln Ala Ser Gly Gln
Pro Pro Pro Thr 50 55 60 atc cgc tgg ttg ctg aat ggg cag ccc ctg
agc atg gtg ccc cca gac 303 Ile Arg Trp Leu Leu Asn Gly Gln Pro Leu
Ser Met Val Pro Pro Asp 65 70 75 cca cac cac ctc ctg cct gat ggg
acc ctt ctg ctg cta cag ccc cct 351 Pro His His Leu Leu Pro Asp Gly
Thr Leu Leu Leu Leu Gln Pro Pro 80 85 90 gcc cgg gga cat gcc cac
gat ggc cag gcc ctg tcc aca gac ctg ggt 399 Ala Arg Gly His Ala His
Asp Gly Gln Ala Leu Ser Thr Asp Leu Gly 95 100 105 110 gtc tac aca
tgt gag gcc agc aac cgg ctt ggc acg gca gtc agc aga 447 Val Tyr Thr
Cys Glu Ala Ser Asn Arg Leu Gly Thr Ala Val Ser Arg 115 120 125 ggc
gct cgg ctg tct gtg gct gtc ctc cgg gag gat ttc cag atc cag 495 Gly
Ala Arg Leu Ser Val Ala Val Leu Arg Glu Asp Phe Gln Ile Gln 130 135
140 cct cgg gac atg gtg gct gtg gtg ggt gag cag ttt act ctg gaa tgt
543 Pro Arg Asp Met Val Ala Val Val Gly Glu Gln Phe Thr Leu Glu Cys
145 150 155 ggg ccg ccc tgg ggc cac cca gag ccc aca gtc tca tgg tgg
aaa gat 591 Gly Pro Pro Trp Gly His Pro Glu Pro Thr Val Ser Trp Trp
Lys Asp 160 165 170 ggg aaa ccc ctg gcc ctc cag ccc gga agg cac aca
gtg tcc ggg ggg 639 Gly Lys Pro Leu Ala Leu Gln Pro Gly Arg His Thr
Val Ser Gly Gly 175 180 185 190 tcc ctg ctg atg gca aga gca gag aag
agt gac gaa ggg acc tac atg 687 Ser Leu Leu Met Ala Arg Ala Glu Lys
Ser Asp Glu Gly Thr Tyr Met 195 200 205 tgt gtg gcc acc aac agc gca
gga cat agg gag agc cgc gca gcc cgg 735 Cys Val Ala Thr Asn Ser Ala
Gly His Arg Glu Ser Arg Ala Ala Arg 210 215 220 gtt tcc atc cag gag
ccc cag gac tac acg gag cct gtg gag ctt ctg 783 Val Ser Ile Gln Glu
Pro Gln Asp Tyr Thr Glu Pro Val Glu Leu Leu 225 230 235 gct gtg cga
att cag ctg gaa aat gtg aca ctg ctg aac ccg gat cct 831 Ala Val Arg
Ile Gln Leu Glu Asn Val Thr Leu Leu Asn Pro Asp Pro 240 245 250 gca
gag ggc ccc aag cct aga ccg gcg gtg tgg ctc agc tgg aag gtc 879 Ala
Glu Gly Pro Lys Pro Arg Pro Ala Val Trp Leu Ser Trp Lys Val 255 260
265 270 agt ggc cct gct gcg cct gcc caa tct tac acg gcc ttg ttc agg
acc 927 Ser Gly Pro Ala Ala Pro Ala Gln Ser Tyr Thr Ala Leu Phe Arg
Thr 275 280 285 cag act gcc ccg gga ggc cag gga gct ccg tgg gca gag
gag ctg ctg 975 Gln Thr Ala Pro Gly Gly Gln Gly Ala Pro Trp Ala Glu
Glu Leu Leu 290 295 300 gcc ggc tgg cag agc gca gag ctt gga ggc ctc
cac tgg ggc caa gac 1023 Ala Gly Trp Gln Ser Ala Glu Leu Gly Gly
Leu His Trp Gly Gln Asp 305 310 315 tac gag ttc aaa gtg aga cca tcc
tct ggc cgg gct cga ggc cct gac 1071 Tyr Glu Phe Lys Val Arg Pro
Ser Ser Gly Arg Ala Arg Gly Pro Asp 320 325 330 agc aac gtg ctg ctc
ctg agg ctg ccg gaa aaa gtg ccc agt gcc cca 1119 Ser Asn Val Leu
Leu Leu Arg Leu Pro Glu Lys Val Pro Ser Ala Pro 335 340 345 350 cct
cag gaa gtg act cta aag cct ggc aat ggc act gtc ttt gtg agc 1167
Pro Gln Glu Val Thr Leu Lys Pro Gly Asn Gly Thr Val Phe Val Ser 355
360 365 tgg gtc cca cca cct gct gaa aac cac aat ggc atc atc cgt ggc
tac 1215 Trp Val Pro Pro Pro Ala Glu Asn His Asn Gly Ile Ile Arg
Gly Tyr 370 375 380 cag gtc tgg agc ctg ggc aac aca tca ctg cca cca
gcc aac tgg act 1263 Gln Val Trp Ser Leu Gly Asn Thr Ser Leu Pro
Pro Ala Asn Trp Thr 385 390 395 gta gtt ggt gag cag acc cag ctg gaa
atc gcc acc cat atg cca ggc 1311 Val Val Gly Glu Gln Thr Gln Leu
Glu Ile Ala Thr His Met Pro Gly 400 405 410 tcc tac tgc gtg caa gtg
gct gca gtc act ggt gct gga gct ggg gag 1359 Ser Tyr Cys Val Gln
Val Ala Ala Val Thr Gly Ala Gly Ala Gly Glu 415 420 425 430 ccc agt
aga cct gtc tgc ctc ctt tta gag cag gcc atg gag cga gcc 1407 Pro
Ser Arg Pro Val Cys Leu Leu Leu Glu Gln Ala Met Glu Arg Ala 435 440
445 acc caa gaa ccc agt gag cat ggt ccc tgg acc ctg gag cag ctg agg
1455 Thr Gln Glu Pro Ser Glu His Gly Pro Trp Thr Leu Glu Gln Leu
Arg 450 455 460 gct acc ttg aag cgg cct gag gtc att gcc acc tgc ggt
gtt gca ctc 1503 Ala Thr Leu Lys Arg Pro Glu Val Ile Ala Thr Cys
Gly Val Ala Leu 465 470 475 tgg ctg ctg ctt ctg ggc acc gcc gtg tgt
atc cac cgc cgg cgc cga 1551 Trp Leu Leu Leu Leu Gly Thr Ala Val
Cys Ile His Arg Arg Arg Arg 480 485 490 gct agg gtg cac ctg ggc cca
ggt ctg tac aga tat acc agt gag gat 1599 Ala Arg Val His Leu Gly
Pro Gly Leu Tyr Arg Tyr Thr Ser Glu Asp 495 500 505 510 gcc atc cta
aaa cac agg atg gat cac agt gac tcc cag tgg ttg gca 1647 Ala Ile
Leu Lys His Arg Met Asp His Ser Asp Ser Gln Trp Leu Ala 515 520 525
gac act tgg cgt tcc acc tct ggc tct cgg gac ctg agc agc agc agc
1695 Asp Thr Trp Arg Ser Thr Ser Gly Ser Arg Asp Leu Ser Ser Ser
Ser 530 535 540 agc ctc agc agt cgg ctg ggg gcg gat gcc cgg gac cca
cta gac tgt 1743 Ser Leu Ser Ser Arg Leu Gly Ala Asp Ala Arg Asp
Pro Leu Asp Cys 545 550 555 cgt cgc tcc ttg ctc tcc tgg gac tcc cga
agc ccc ggc gtg ccc ctg 1791 Arg Arg Ser Leu Leu Ser Trp Asp Ser
Arg Ser Pro Gly Val Pro Leu 560 565 570 ctt cca gac acc agc act ttt
tat ggc tcc ctc atc gct gag ctg ccc 1839 Leu Pro Asp Thr Ser Thr
Phe Tyr Gly Ser Leu Ile Ala Glu Leu Pro 575 580 585 590 tcc agt acc
cca gcc agg cca agt ccc cag gtc cca gct gtc agg cgc 1887 Ser Ser
Thr Pro Ala Arg Pro Ser Pro Gln Val Pro Ala Val Arg Arg 595 600 605
ctc cca ccc cag ctg gcc cag ctc tcc agc ccc tgt tcc agc tca gac
1935 Leu Pro Pro Gln Leu Ala Gln Leu Ser Ser Pro Cys Ser Ser Ser
Asp 610 615 620 agc ctc tgc agc cgc agg gga ctc tct tct ccc cgc ttg
tct ctg gcc 1983 Ser Leu Cys Ser Arg Arg Gly Leu Ser Ser Pro Arg
Leu Ser Leu Ala 625 630 635 cct gca gag gct tgg aag gcc aaa aag aag
cag gag ctg cag cat gcc 2031 Pro Ala Glu Ala Trp Lys Ala Lys Lys
Lys Gln Glu Leu Gln His Ala 640 645 650 aac agt tcc cca ctg ctc cgg
ggc agc cac tcc ttg gag ctc cgg gcc 2079 Asn Ser Ser Pro Leu Leu
Arg Gly Ser His Ser Leu Glu Leu Arg Ala 655 660 665 670 tgt gag tta
gga aat aga ggt tcc aag aac ctt tcc caa agc cca gga 2127 Cys Glu
Leu Gly Asn Arg Gly Ser Lys Asn Leu Ser Gln Ser Pro Gly 675 680 685
gct gtg ccc caa gct ctg gtt gcc tgg cgg gcc ctg gga ccg aaa ctc
2175 Ala Val Pro Gln Ala Leu Val Ala Trp Arg Ala Leu Gly Pro Lys
Leu 690 695 700 ctc agc tcc tca aat gag ctg gtt act cgt cat ctc cct
cca gca ccc 2223 Leu Ser Ser Ser Asn Glu Leu Val Thr Arg His Leu
Pro Pro Ala Pro 705 710 715 ctc ttt cct cat gaa act ccc cca act cag
agt caa cag acc cag cct 2271 Leu Phe Pro His Glu Thr Pro Pro Thr
Gln Ser Gln Gln Thr Gln Pro 720 725 730 ccg gtg gca cca cag gct ccc
tcc tcc atc ctg ctg cca gca gcc ccc 2319 Pro Val Ala Pro Gln Ala
Pro Ser Ser Ile Leu Leu Pro Ala Ala Pro 735 740 745 750 atc ccc atc
ctt agc ccc tgc agt ccc cct agc ccc cag gcc tct tcc 2367 Ile Pro
Ile Leu Ser Pro Cys Ser Pro Pro Ser Pro Gln Ala Ser Ser 755 760 765
ctc tct ggc ccc agc cca gct tcc agt cgc ctg tcc agc tcc tca ctg
2415 Leu Ser Gly Pro Ser Pro Ala Ser Ser Arg Leu Ser Ser Ser Ser
Leu 770 775 780 tca tcc ctg ggg gag gat caa gac agc gtg ctg acc cct
gag gag gta 2463 Ser Ser Leu Gly Glu Asp Gln Asp Ser Val Leu Thr
Pro Glu Glu Val 785 790 795 gcc ctg tgc ttg gaa ctc agt gag ggt gag
gag act ccc agg aac agc 2511 Ala Leu Cys Leu Glu Leu Ser Glu Gly
Glu Glu Thr Pro Arg Asn Ser 800 805 810 gtc tct ccc atg cca agg gct
cct tca ccc ccc acc acc tat ggg tac 2559 Val Ser Pro Met Pro Arg
Ala Pro Ser Pro Pro Thr Thr Tyr Gly Tyr 815 820 825 830 atc agc gtc
cca aca gcc tca gag ttc acg gac atg ggc agg act gga 2607 Ile Ser
Val Pro Thr Ala Ser Glu Phe Thr Asp Met Gly Arg Thr Gly 835 840 845
gga ggg gtg ggg ccc aag ggg gga gtc ttg ctg tgc cca cct cgg ccc
2655 Gly Gly Val Gly Pro Lys Gly Gly Val Leu Leu Cys Pro Pro Arg
Pro 850 855 860 tgc ctc acc ccc acc ccc agc gag ggc tcc tta gcc aat
ggt tgg ggc 2703 Cys Leu Thr Pro Thr Pro Ser Glu Gly Ser Leu Ala
Asn Gly Trp Gly 865 870 875 tca gcc tct gag gac aat gcc gcc agc gcc
aga gcc agc ctt gtc agc 2751 Ser Ala Ser Glu Asp Asn Ala Ala Ser
Ala Arg Ala Ser Leu Val Ser 880 885 890 tcc tcc gat ggc tcc ttc ctc
gct gat gct cac ttt gcc cgg gcc ctg 2799 Ser Ser Asp Gly Ser Phe
Leu Ala Asp Ala His Phe Ala Arg Ala Leu 895 900 905 910 gca gtg gct
gtg gat agc ttt ggt ttc ggt cta gag ccc agg gag gca 2847 Ala Val
Ala Val Asp Ser Phe Gly Phe Gly Leu Glu Pro Arg Glu Ala 915 920 925
gac tgc gtc ttc ata gat gcc tca tca cct ccc tcc cca cgg gat gag
2895 Asp Cys Val Phe Ile Asp Ala Ser Ser Pro Pro Ser Pro Arg Asp
Glu 930 935 940 atc ttc ctg acc ccc aac ctc tcc ctg ccc ctg tgg gag
tgg agg cca 2943 Ile Phe Leu Thr Pro Asn Leu Ser Leu Pro Leu Trp
Glu Trp Arg Pro 945 950 955 gac tgg ttg gaa gac atg gag gtc agc cac
acc cag cgg ctg gga agg 2991 Asp Trp Leu Glu Asp Met Glu Val Ser
His Thr Gln Arg Leu Gly Arg 960 965 970 ggg atg cct ccc tgg ccc cct
gaa ctc tca gat ctc ttc cca gag aag 3039 Gly Met Pro Pro Trp Pro
Pro Glu Leu Ser Asp Leu Phe Pro Glu Lys 975 980 985 990 tca gct cca
ctg tcg tat gcc caa ggc tgg tgc ttc tcc tgt aga tta 3087 Ser Ala
Pro Leu Ser Tyr Ala Gln Gly Trp Cys Phe Ser Cys Arg Leu 995 1000
1005 ctc ctg aac cgt gtc cct gag act tcc cag acg gga atc aga acc
act 3135 Leu Leu Asn Arg Val Pro Glu Thr Ser Gln Thr Gly Ile Arg
Thr Thr 1010 1015 1020 tct cct gtt cca ccc aca aga cct ggg ctg tgg
tgt gtg ggt ctt ggc 3183 Ser Pro Val Pro Pro Thr Arg Pro Gly Leu
Trp Cys Val Gly Leu Gly 1025 1030 1035 ctg tgt ttc tct gca gct ggg
gtc cac ctt ccc aag cct cca gag agt 3231 Leu Cys Phe Ser Ala Ala
Gly Val His Leu Pro Lys Pro Pro Glu Ser 1040 1045 1050 tct ccc tcc
acg att gtg aaa aca aat gaa aac aaa att aga gca aag 3279 Ser Pro
Ser Thr Ile Val Lys Thr Asn Glu Asn Lys Ile Arg Ala Lys 1055 1060
1065 1070 ctg acc tgg agc cct cag gga gca aaa cat cat ctc cac ctg
act cct 3327 Leu Thr Trp Ser Pro Gln Gly Ala Lys His His Leu His
Leu Thr Pro 1075 1080 1085 agc cac tgc ttt ctc ctc tgt gcc atc cac
tcc cac cac cag gtt gtt 3375 Ser His Cys Phe Leu Leu Cys Ala Ile
His Ser His His Gln Val Val 1090 1095 1100 ttg gcc tgaggagcag
ccctgcctgc tgctcttccc ccaccatttg gatcacagga 3431 Leu Ala agtggaggag
ccagaggtgc ctttgtggag gacagcagtg gctgctggga gagggctgtg 3491
gaggaaggag cttctcggag ccccctctca gccttacctg ggcccctcct ctagagaaga
3551 gctcaactct ctcccaacct caccatggaa agaaaataat tatgaatgcc
actgaggcac 3611 tgaggcccta cctcatgcca aacaaagggt tcaaggctgg
gtctagcgag gatgctgaag 3671 gaagggaggt atgagacccg taggtcaaaa
gcaccatcct cgta 3715 37 1104 PRT Homo sapiens 37 Met Gly Ser Gly
Gly Asp Ser Leu Leu Gly Gly Arg Gly Ser Leu Pro 1 5 10 15 Leu Leu
Leu Leu Leu Ile Met Gly Gly Met Ala Gln Asp Ser Pro Pro 20 25 30
Gln Ile Leu Val His Pro Gln Asp Gln Leu Phe Gln Gly Pro Gly Pro 35
40 45 Ala Arg Met Ser Cys Gln Ala Ser Gly Gln Pro Pro Pro Thr Ile
Arg 50 55 60 Trp Leu Leu Asn Gly Gln Pro Leu Ser Met Val Pro Pro
Asp Pro His 65 70 75 80 His Leu Leu Pro Asp Gly Thr Leu Leu Leu Leu
Gln Pro Pro Ala Arg 85 90 95 Gly His Ala His Asp Gly Gln Ala Leu
Ser Thr Asp Leu Gly Val Tyr 100 105 110 Thr Cys Glu Ala Ser Asn Arg
Leu Gly Thr Ala Val Ser Arg Gly Ala 115 120 125 Arg Leu Ser Val Ala
Val Leu Arg Glu Asp Phe Gln Ile Gln Pro Arg 130 135 140 Asp Met Val
Ala Val Val Gly Glu Gln Phe Thr Leu Glu Cys Gly Pro 145 150 155 160
Pro Trp Gly His Pro Glu Pro Thr Val Ser Trp Trp Lys Asp Gly Lys 165
170 175 Pro Leu Ala Leu Gln Pro Gly Arg His Thr Val Ser Gly Gly Ser
Leu 180 185 190 Leu Met Ala Arg Ala Glu Lys Ser Asp Glu Gly Thr Tyr
Met Cys Val 195 200 205 Ala Thr Asn Ser Ala Gly His Arg Glu Ser Arg
Ala Ala Arg Val Ser 210 215 220 Ile Gln Glu Pro Gln Asp Tyr Thr Glu
Pro Val Glu Leu Leu Ala Val 225 230 235 240 Arg Ile Gln Leu Glu Asn
Val Thr Leu Leu Asn Pro Asp Pro Ala Glu 245 250 255 Gly Pro Lys Pro
Arg Pro Ala Val Trp Leu Ser Trp Lys Val Ser Gly 260 265 270 Pro Ala
Ala Pro Ala Gln Ser Tyr Thr Ala Leu Phe Arg Thr Gln Thr 275 280 285
Ala Pro Gly Gly Gln Gly Ala Pro Trp Ala Glu Glu Leu Leu Ala Gly 290
295 300 Trp Gln Ser Ala Glu Leu Gly Gly Leu His Trp Gly Gln Asp Tyr
Glu 305 310 315 320 Phe Lys Val Arg Pro Ser Ser Gly Arg Ala Arg Gly
Pro Asp Ser Asn 325 330 335 Val Leu Leu Leu Arg Leu Pro Glu Lys Val
Pro Ser Ala Pro Pro Gln 340 345 350 Glu Val Thr Leu Lys Pro Gly Asn
Gly Thr Val Phe Val Ser Trp Val 355 360 365 Pro Pro Pro Ala Glu Asn
His Asn Gly Ile Ile Arg Gly Tyr Gln Val 370 375 380 Trp Ser Leu Gly
Asn Thr Ser Leu Pro Pro Ala Asn Trp Thr Val Val 385 390 395 400 Gly
Glu Gln Thr Gln Leu Glu Ile Ala Thr His Met Pro Gly Ser Tyr 405 410
415 Cys Val Gln Val Ala Ala Val Thr Gly Ala Gly Ala Gly Glu Pro Ser
420 425 430 Arg Pro Val Cys Leu Leu Leu Glu Gln Ala Met Glu Arg Ala
Thr Gln 435
440 445 Glu Pro Ser Glu His Gly Pro Trp Thr Leu Glu Gln Leu Arg Ala
Thr 450 455 460 Leu Lys Arg Pro Glu Val Ile Ala Thr Cys Gly Val Ala
Leu Trp Leu 465 470 475 480 Leu Leu Leu Gly Thr Ala Val Cys Ile His
Arg Arg Arg Arg Ala Arg 485 490 495 Val His Leu Gly Pro Gly Leu Tyr
Arg Tyr Thr Ser Glu Asp Ala Ile 500 505 510 Leu Lys His Arg Met Asp
His Ser Asp Ser Gln Trp Leu Ala Asp Thr 515 520 525 Trp Arg Ser Thr
Ser Gly Ser Arg Asp Leu Ser Ser Ser Ser Ser Leu 530 535 540 Ser Ser
Arg Leu Gly Ala Asp Ala Arg Asp Pro Leu Asp Cys Arg Arg 545 550 555
560 Ser Leu Leu Ser Trp Asp Ser Arg Ser Pro Gly Val Pro Leu Leu Pro
565 570 575 Asp Thr Ser Thr Phe Tyr Gly Ser Leu Ile Ala Glu Leu Pro
Ser Ser 580 585 590 Thr Pro Ala Arg Pro Ser Pro Gln Val Pro Ala Val
Arg Arg Leu Pro 595 600 605 Pro Gln Leu Ala Gln Leu Ser Ser Pro Cys
Ser Ser Ser Asp Ser Leu 610 615 620 Cys Ser Arg Arg Gly Leu Ser Ser
Pro Arg Leu Ser Leu Ala Pro Ala 625 630 635 640 Glu Ala Trp Lys Ala
Lys Lys Lys Gln Glu Leu Gln His Ala Asn Ser 645 650 655 Ser Pro Leu
Leu Arg Gly Ser His Ser Leu Glu Leu Arg Ala Cys Glu 660 665 670 Leu
Gly Asn Arg Gly Ser Lys Asn Leu Ser Gln Ser Pro Gly Ala Val 675 680
685 Pro Gln Ala Leu Val Ala Trp Arg Ala Leu Gly Pro Lys Leu Leu Ser
690 695 700 Ser Ser Asn Glu Leu Val Thr Arg His Leu Pro Pro Ala Pro
Leu Phe 705 710 715 720 Pro His Glu Thr Pro Pro Thr Gln Ser Gln Gln
Thr Gln Pro Pro Val 725 730 735 Ala Pro Gln Ala Pro Ser Ser Ile Leu
Leu Pro Ala Ala Pro Ile Pro 740 745 750 Ile Leu Ser Pro Cys Ser Pro
Pro Ser Pro Gln Ala Ser Ser Leu Ser 755 760 765 Gly Pro Ser Pro Ala
Ser Ser Arg Leu Ser Ser Ser Ser Leu Ser Ser 770 775 780 Leu Gly Glu
Asp Gln Asp Ser Val Leu Thr Pro Glu Glu Val Ala Leu 785 790 795 800
Cys Leu Glu Leu Ser Glu Gly Glu Glu Thr Pro Arg Asn Ser Val Ser 805
810 815 Pro Met Pro Arg Ala Pro Ser Pro Pro Thr Thr Tyr Gly Tyr Ile
Ser 820 825 830 Val Pro Thr Ala Ser Glu Phe Thr Asp Met Gly Arg Thr
Gly Gly Gly 835 840 845 Val Gly Pro Lys Gly Gly Val Leu Leu Cys Pro
Pro Arg Pro Cys Leu 850 855 860 Thr Pro Thr Pro Ser Glu Gly Ser Leu
Ala Asn Gly Trp Gly Ser Ala 865 870 875 880 Ser Glu Asp Asn Ala Ala
Ser Ala Arg Ala Ser Leu Val Ser Ser Ser 885 890 895 Asp Gly Ser Phe
Leu Ala Asp Ala His Phe Ala Arg Ala Leu Ala Val 900 905 910 Ala Val
Asp Ser Phe Gly Phe Gly Leu Glu Pro Arg Glu Ala Asp Cys 915 920 925
Val Phe Ile Asp Ala Ser Ser Pro Pro Ser Pro Arg Asp Glu Ile Phe 930
935 940 Leu Thr Pro Asn Leu Ser Leu Pro Leu Trp Glu Trp Arg Pro Asp
Trp 945 950 955 960 Leu Glu Asp Met Glu Val Ser His Thr Gln Arg Leu
Gly Arg Gly Met 965 970 975 Pro Pro Trp Pro Pro Glu Leu Ser Asp Leu
Phe Pro Glu Lys Ser Ala 980 985 990 Pro Leu Ser Tyr Ala Gln Gly Trp
Cys Phe Ser Cys Arg Leu Leu Leu 995 1000 1005 Asn Arg Val Pro Glu
Thr Ser Gln Thr Gly Ile Arg Thr Thr Ser Pro 1010 1015 1020 Val Pro
Pro Thr Arg Pro Gly Leu Trp Cys Val Gly Leu Gly Leu Cys 1025 1030
1035 1040 Phe Ser Ala Ala Gly Val His Leu Pro Lys Pro Pro Glu Ser
Ser Pro 1045 1050 1055 Ser Thr Ile Val Lys Thr Asn Glu Asn Lys Ile
Arg Ala Lys Leu Thr 1060 1065 1070 Trp Ser Pro Gln Gly Ala Lys His
His Leu His Leu Thr Pro Ser His 1075 1080 1085 Cys Phe Leu Leu Cys
Ala Ile His Ser His His Gln Val Val Leu Ala 1090 1095 1100 38 3688
DNA Mus musculus CDS (6)...(3050) CDS (3393)...(3509) CDS
(3579)...(3680) 38 agtgt atg gga caa gga gag gag ccg aga gca gcc
atg ggc tct gga gga 50 Met Gly Gln Gly Glu Glu Pro Arg Ala Ala Met
Gly Ser Gly Gly 1 5 10 15 acg ggc ctc ctg ggg acg gag tgg cct ctg
cct ctg ctg ctg ctt ttc 98 Thr Gly Leu Leu Gly Thr Glu Trp Pro Leu
Pro Leu Leu Leu Leu Phe 20 25 30 atc atg gga ggt gag gct ctg gat
tct cca ccc cag atc cta gtt cac 146 Ile Met Gly Gly Glu Ala Leu Asp
Ser Pro Pro Gln Ile Leu Val His 35 40 45 ccc cag gac cag cta ctt
cag ggc tct ggc cca gcc aag atg agg tgc 194 Pro Gln Asp Gln Leu Leu
Gln Gly Ser Gly Pro Ala Lys Met Arg Cys 50 55 60 aga tca tcc ggc
caa cca cct ccc act atc cgc tgg ctg ctg aat ggg 242 Arg Ser Ser Gly
Gln Pro Pro Pro Thr Ile Arg Trp Leu Leu Asn Gly 65 70 75 cag ccc
ctc agc atg gcc acc cca gac cta cat tac ctt ttg ccg gat 290 Gln Pro
Leu Ser Met Ala Thr Pro Asp Leu His Tyr Leu Leu Pro Asp 80 85 90 95
ggg acc ctc ctg tta cat cgg ccc tct gtc cag gga cgg cca caa gat 338
Gly Thr Leu Leu Leu His Arg Pro Ser Val Gln Gly Arg Pro Gln Asp 100
105 110 gac cag aac atc ctc tca gca atc ctg ggt gtc tac aca tgt gag
gcc 386 Asp Gln Asn Ile Leu Ser Ala Ile Leu Gly Val Tyr Thr Cys Glu
Ala 115 120 125 agc aac cgg ctg ggc aca gca gtg agc cgg ggt gct agg
ctg tct gtg 434 Ser Asn Arg Leu Gly Thr Ala Val Ser Arg Gly Ala Arg
Leu Ser Val 130 135 140 gct gtc ctc cag gag gac ttc cag atc caa cct
cgg gac aca gtg gcc 482 Ala Val Leu Gln Glu Asp Phe Gln Ile Gln Pro
Arg Asp Thr Val Ala 145 150 155 gtg gtg gga gag agc ttg gtt ctt gag
tgt ggt cct ccc tgg ggc tac 530 Val Val Gly Glu Ser Leu Val Leu Glu
Cys Gly Pro Pro Trp Gly Tyr 160 165 170 175 cca aaa ccc tcg gtc tca
tgg tgg aaa gac ggg aaa ccc ctg gtc ctc 578 Pro Lys Pro Ser Val Ser
Trp Trp Lys Asp Gly Lys Pro Leu Val Leu 180 185 190 cag cca ggg agg
cgc aca gta tct ggg gat tcc ctg atg gtg tca aga 626 Gln Pro Gly Arg
Arg Thr Val Ser Gly Asp Ser Leu Met Val Ser Arg 195 200 205 gca gag
aag aat gac tcg ggg acc tat atg tgt atg gcc acc aac aat 674 Ala Glu
Lys Asn Asp Ser Gly Thr Tyr Met Cys Met Ala Thr Asn Asn 210 215 220
gct ggg caa cgg gag agc cga gca gcc agg gtg tct atc cag gaa tcc 722
Ala Gly Gln Arg Glu Ser Arg Ala Ala Arg Val Ser Ile Gln Glu Ser 225
230 235 cag gac cac aag gaa cat cta gag ctt ctg gct gtt cgc att cag
ctg 770 Gln Asp His Lys Glu His Leu Glu Leu Leu Ala Val Arg Ile Gln
Leu 240 245 250 255 gaa aat gtg acc ctg cta aac ccc gaa cct gta aaa
ggt ccc aag cct 818 Glu Asn Val Thr Leu Leu Asn Pro Glu Pro Val Lys
Gly Pro Lys Pro 260 265 270 ggg cca tcc gtg tgg ctc agc tgg aag gtg
agc ggc cct gct gca cct 866 Gly Pro Ser Val Trp Leu Ser Trp Lys Val
Ser Gly Pro Ala Ala Pro 275 280 285 gct gag tca tac aca gct ctg ttc
agg act cag agg tcc ccc agg gac 914 Ala Glu Ser Tyr Thr Ala Leu Phe
Arg Thr Gln Arg Ser Pro Arg Asp 290 295 300 caa gga tct cca tgg aca
gag gtg ctg ctg cgt ggc ttg cag agt gca 962 Gln Gly Ser Pro Trp Thr
Glu Val Leu Leu Arg Gly Leu Gln Ser Ala 305 310 315 aag ctt ggg ggt
ctc cac tgg ggc caa gac tat gaa ttc aaa gtg aga 1010 Lys Leu Gly
Gly Leu His Trp Gly Gln Asp Tyr Glu Phe Lys Val Arg 320 325 330 335
ccg tcc tcc ggc cgg gct cga ggc cct gac agc aat gtg ttg ctc ctg
1058 Pro Ser Ser Gly Arg Ala Arg Gly Pro Asp Ser Asn Val Leu Leu
Leu 340 345 350 agg ctg cct gaa cag gtg ccc agt gcc cca cct caa gga
gtg acc tta 1106 Arg Leu Pro Glu Gln Val Pro Ser Ala Pro Pro Gln
Gly Val Thr Leu 355 360 365 aga tct ggc aac ggt agt gtc ttt gtg agt
tgg gct cca cca cct gct 1154 Arg Ser Gly Asn Gly Ser Val Phe Val
Ser Trp Ala Pro Pro Pro Ala 370 375 380 gaa agc cat aat ggt gtc atc
cgt ggt tac cag gtc tgg agc ctg ggc 1202 Glu Ser His Asn Gly Val
Ile Arg Gly Tyr Gln Val Trp Ser Leu Gly 385 390 395 aat gcc tca ttg
cct gct gcc aac tgg acc gta gtg ggt gaa cag acc 1250 Asn Ala Ser
Leu Pro Ala Ala Asn Trp Thr Val Val Gly Glu Gln Thr 400 405 410 415
cag ctg gag atc gcc aca cga ctg cca ggc tcc tat tgt gtg caa gtg
1298 Gln Leu Glu Ile Ala Thr Arg Leu Pro Gly Ser Tyr Cys Val Gln
Val 420 425 430 gct gca gtc act gga gct ggt gct gga gaa ctc agt acc
cct gtc tgc 1346 Ala Ala Val Thr Gly Ala Gly Ala Gly Glu Leu Ser
Thr Pro Val Cys 435 440 445 ctc ctt tta gag cag gcc atg gag caa tca
gca cga gac ccc agg aaa 1394 Leu Leu Leu Glu Gln Ala Met Glu Gln
Ser Ala Arg Asp Pro Arg Lys 450 455 460 cat gtt ccc tgg acc ctg gaa
cag ctg agg gcc acc ttg aga cga cca 1442 His Val Pro Trp Thr Leu
Glu Gln Leu Arg Ala Thr Leu Arg Arg Pro 465 470 475 gaa gtc att gcc
agt agt gct gtc cta ctc tgg ttg ctg cta cta ggc 1490 Glu Val Ile
Ala Ser Ser Ala Val Leu Leu Trp Leu Leu Leu Leu Gly 480 485 490 495
att act gtg tgt atc tac aga cga cgc aaa gct ggg gtg cac ctg ggc
1538 Ile Thr Val Cys Ile Tyr Arg Arg Arg Lys Ala Gly Val His Leu
Gly 500 505 510 cca ggt ctg tac aga tac acc agc gag gac gcc att cta
aaa cac agg 1586 Pro Gly Leu Tyr Arg Tyr Thr Ser Glu Asp Ala Ile
Leu Lys His Arg 515 520 525 atg gac cac agt gac tcc cca tgg ctg gca
gac acc tgg cgt tcc acc 1634 Met Asp His Ser Asp Ser Pro Trp Leu
Ala Asp Thr Trp Arg Ser Thr 530 535 540 tct ggc tct cga gac ctg agc
agc agc agc agc ctt agt agt cgg ctg 1682 Ser Gly Ser Arg Asp Leu
Ser Ser Ser Ser Ser Leu Ser Ser Arg Leu 545 550 555 gga ttg gac cct
cgg gac cca cta gag ggc agg cgc tcc ttg atc tcc 1730 Gly Leu Asp
Pro Arg Asp Pro Leu Glu Gly Arg Arg Ser Leu Ile Ser 560 565 570 575
tgg gac cct cgg agc ccc ggt gta ccc ctg ctt cca gac acg agc acg
1778 Trp Asp Pro Arg Ser Pro Gly Val Pro Leu Leu Pro Asp Thr Ser
Thr 580 585 590 ttt tac ggc tcc ctc att gca gag cag cct tcc agc cct
cca gtc cgg 1826 Phe Tyr Gly Ser Leu Ile Ala Glu Gln Pro Ser Ser
Pro Pro Val Arg 595 600 605 cca agc ccc aag aca cca gct gct agg cgc
ttt cca tcc aag ttg gct 1874 Pro Ser Pro Lys Thr Pro Ala Ala Arg
Arg Phe Pro Ser Lys Leu Ala 610 615 620 gga acc tcc agc ccc tgg gct
agc tca gat agt ctc tgc agc cgc agg 1922 Gly Thr Ser Ser Pro Trp
Ala Ser Ser Asp Ser Leu Cys Ser Arg Arg 625 630 635 gga ctc tgt tcc
cca cgc atg tct ctg acc cct aca gag gct tgg aag 1970 Gly Leu Cys
Ser Pro Arg Met Ser Leu Thr Pro Thr Glu Ala Trp Lys 640 645 650 655
gcc aaa aag aag cag gaa ttg cac caa gct aac agc tcc cca ctg ctc
2018 Ala Lys Lys Lys Gln Glu Leu His Gln Ala Asn Ser Ser Pro Leu
Leu 660 665 670 cgg ggc agc cac ccc atg gaa atc tgg gcc tgg gag ttg
gga agc aga 2066 Arg Gly Ser His Pro Met Glu Ile Trp Ala Trp Glu
Leu Gly Ser Arg 675 680 685 gcc tcc aag aac ctt tct caa agc cca gga
gaa gcg ccc cga gcc gtg 2114 Ala Ser Lys Asn Leu Ser Gln Ser Pro
Gly Glu Ala Pro Arg Ala Val 690 695 700 gta tcc tgg cgt gct gtg gga
cca caa ctt cac cgc aac tcc agt gag 2162 Val Ser Trp Arg Ala Val
Gly Pro Gln Leu His Arg Asn Ser Ser Glu 705 710 715 ctg gca tct cgt
cca ctc cct cca aca ccc ctt tct ctt cgt gga gct 2210 Leu Ala Ser
Arg Pro Leu Pro Pro Thr Pro Leu Ser Leu Arg Gly Ala 720 725 730 735
tcc agt cat gac cca cag agc cag tgt gtg gag aag ctc caa gct ccc
2258 Ser Ser His Asp Pro Gln Ser Gln Cys Val Glu Lys Leu Gln Ala
Pro 740 745 750 tcc tct gac cca ctg cca gca gcc cct ctc tcc gtc ctc
aac tct tcc 2306 Ser Ser Asp Pro Leu Pro Ala Ala Pro Leu Ser Val
Leu Asn Ser Ser 755 760 765 aga cct tcc agc ccc cag gcc tct ttc ctc
tcc tgt cct agc cca tcc 2354 Arg Pro Ser Ser Pro Gln Ala Ser Phe
Leu Ser Cys Pro Ser Pro Ser 770 775 780 tcc agc aac ctg tcc agc tcc
tcg ctg tca tcc tta gag gag gag gag 2402 Ser Ser Asn Leu Ser Ser
Ser Ser Leu Ser Ser Leu Glu Glu Glu Glu 785 790 795 gat cag gac agc
gtg ctc acc ccc gag gag gta gcc ctg tgt ctg gag 2450 Asp Gln Asp
Ser Val Leu Thr Pro Glu Glu Val Ala Leu Cys Leu Glu 800 805 810 815
ctc agt gat ggg gag gag aca ccc acg aac agt gta tct cct atg cca
2498 Leu Ser Asp Gly Glu Glu Thr Pro Thr Asn Ser Val Ser Pro Met
Pro 820 825 830 aga gct cct tcc ccg cca aca acc tat ggc tat atc agc
ata cca acc 2546 Arg Ala Pro Ser Pro Pro Thr Thr Tyr Gly Tyr Ile
Ser Ile Pro Thr 835 840 845 tgc tca gga ctg gca gac atg ggc aga gct
ggc ggg ggc gtg ggg tct 2594 Cys Ser Gly Leu Ala Asp Met Gly Arg
Ala Gly Gly Gly Val Gly Ser 850 855 860 gag gtt ggg aac tta ctg tat
cca cct cgg ccc tgc ccc acc cct aca 2642 Glu Val Gly Asn Leu Leu
Tyr Pro Pro Arg Pro Cys Pro Thr Pro Thr 865 870 875 ccc agc gag ggc
tcc ctg gcc aat ggt tgg ggc tca gct tct gag gac 2690 Pro Ser Glu
Gly Ser Leu Ala Asn Gly Trp Gly Ser Ala Ser Glu Asp 880 885 890 895
aat gtc ccc agc gcc agg gcc agc ctg gtt agc tct tct gat ggc tcc
2738 Asn Val Pro Ser Ala Arg Ala Ser Leu Val Ser Ser Ser Asp Gly
Ser 900 905 910 ttc ctc gct gat act cac ttt gct cgt gcc ctg gca gtg
gct gtg gat 2786 Phe Leu Ala Asp Thr His Phe Ala Arg Ala Leu Ala
Val Ala Val Asp 915 920 925 agc ttt ggc ctc agt ctg gat ccc agg gaa
gct gac tgt gtc ttc act 2834 Ser Phe Gly Leu Ser Leu Asp Pro Arg
Glu Ala Asp Cys Val Phe Thr 930 935 940 gat gcc tca tca cct ccc tcc
cct cgg ggt gat ctc tcc ctg acc cga 2882 Asp Ala Ser Ser Pro Pro
Ser Pro Arg Gly Asp Leu Ser Leu Thr Arg 945 950 955 agc ttc tct ctg
cct ttg tgg gag tgg agg cca gac tgg ttg gaa gat 2930 Ser Phe Ser
Leu Pro Leu Trp Glu Trp Arg Pro Asp Trp Leu Glu Asp 960 965 970 975
gct gag atc agc cac acc cag agg ctg ggg agg ggg ctg cct ccc tgg
2978 Ala Glu Ile Ser His Thr Gln Arg Leu Gly Arg Gly Leu Pro Pro
Trp 980 985 990 cct cct gat tct agg gcc tct tcc cag cga agt tgg cta
act ggt gct 3026 Pro Pro Asp Ser Arg Ala Ser Ser Gln Arg Ser Trp
Leu Thr Gly Ala 995 1000 1005 gtg ccc aag gct ggt gat tcc tcc
tgaattgtcc ctgagaaggc cagaagagca 3080 Val Pro Lys Ala Gly Asp Ser
Ser 1010 1015 cccagaccac tctcctgtct gtcccctggc tttctcacat
gtggaggtct tggcctatgc 3140 ttctctgtaa tagaagtcca ccgtcactag
gcttctggag agctctgtca ttgggattgt 3200 taaaataaat gaaagcaaac
caaaatatga tcacgggagt cttggattcc cactgagaac 3260 aagacagcat
cttcaggaca gcagactctc cacaaccaga acctttggcc taagtaagcc 3320
tggctccgga gctcccacct aagtggatca tggaaagaag ggaagccaac caggtcttca
3380 ggaaggacag aa atg ttt ttt ggt gag ggc tat ggt gga gga cct gtg
gaa 3431 Met Phe Phe Gly Glu Gly Tyr Gly Gly Gly Pro Val Glu 1020
1025 gag ccc tct cat atc tac ttg gac tcc tcc ctt aga ggc cag ctc
aac 3479 Glu Pro Ser His Ile Tyr Leu Asp Ser Ser Leu Arg Gly Gln
Leu Asn 1030 1035 1040 cct ttc ccc agt cac acc atg caa gga aac
taaaggagaa aggtcgtgga 3529 Pro Phe Pro Ser His Thr Met Gln Gly Asn
1045 1050 tgcagtgggc cctatacagc gtcacagtca atgcttcaaa gtgagatca atg
gag gag 3587
Met Glu Glu 1055 act gaa gga aag gac gca ggg aaa cag gga acc aat
gcg cta ttc tca 3635 Thr Glu Gly Lys Asp Ala Gly Lys Gln Gly Thr
Asn Ala Leu Phe Ser 1060 1065 1070 ttc tac cgc cac tct gag ctt aag
gaa ctt aat tct ata aaa ctg 3680 Phe Tyr Arg His Ser Glu Leu Lys
Glu Leu Asn Ser Ile Lys Leu 1075 1080 1085 taaagacg 3688 39 3688
DNA Mus musculus 39 tcacataccc tgttcctctc ctcggctctc gtcggtaccc
gagacctcct tgcccggagg 60 acccctgcct caccggagac ggagacgacg
acgaaaagta gtaccctcca ctccgagacc 120 taagaggtgg ggtctaggat
caagtggggg tcctggtcga tgaagtcccg agaccgggtc 180 ggttctactc
cacgtctagt aggccggttg gtggagggtg ataggcgacc gacgacttac 240
ccgtcgggga gtcgtaccgg tggggtctgg atgtaatgga aaacggccta ccctgggagg
300 acaatgtagc cgggagacag gtccctgccg gtgttctact ggtcttgtag
gagagtcgtt 360 aggacccaca gatgtgtaca ctccggtcgt tggccgaccc
gtgtcgtcac tcggccccac 420 gatccgacag acaccgacag gaggtcctcc
tgaaggtcta ggttggagcc ctgtgtcacc 480 ggcaccaccc tctctcgaac
caagaactca caccaggagg gaccccgatg ggttttggga 540 gccagagtac
cacctttctg ccctttgggg accaggaggt cggtccctcc gcgtgtcata 600
gacccctaag ggactaccac agttctcgtc tcttcttact gagcccctgg atatacacat
660 accggtggtt gttacgaccc gttgccctct cggctcgtcg gtcccacaga
taggtcctta 720 gggtcctggt gttccttgta gatctcgaag accgacaagc
gtaagtcgac cttttacact 780 gggacgattt ggggcttgga cattttccag
ggttcggacc cggtaggcac accgagtcga 840 ccttccactc gccgggacga
cgtggacgac tcagtatgtg tcgagacaag tcctgagtct 900 ccagggggtc
cctggttcct agaggtacct gtctccacga cgacgcaccg aacgtctcac 960
gtttcgaacc cccagaggtg accccggttc tgatacttaa gtttcactct ggcaggaggc
1020 cggcccgagc tccgggactg tcgttacaca acgaggactc cgacggactt
gtccacgggt 1080 cacggggtgg agttcctcac tggaattcta gaccgttgcc
atcacagaaa cactcaaccc 1140 gaggtggtgg acgactttcg gtattaccac
agtaggcacc aatggtccag acctcggacc 1200 cgttacggag taacggacga
cggttgacct ggcatcaccc acttgtctgg gtcgacctct 1260 agcggtgtgc
tgacggtccg aggataacac acgttcaccg acgtcagtga cctcgaccac 1320
gacctcttga gtcatgggga cagacggagg aaaatctcgt ccggtacctc gttagtcgtg
1380 ctctggggtc ctttgtacaa gggacctggg accttgtcga ctcccggtgg
aactctgctg 1440 gtcttcagta acggtcatca cgacaggatg agaccaacga
cgatgatccg taatgacaca 1500 catagatgtc tgctgcgttt cgaccccacg
tggacccggg tccagacatg tctatgtggt 1560 cgctcctgcg gtaagatttt
gtgtcctacc tggtgtcact gaggggtacc gaccgtctgt 1620 ggaccgcaag
gtggagaccg agagctctgg actcgtcgtc gtcgtcggaa tcatcagccg 1680
accctaacct gggagccctg ggtgatctcc cgtccgcgag gaactagagg accctgggag
1740 cctcggggcc acatggggac gaaggtctgt gctcgtgcaa aatgccgagg
gagtaacgtc 1800 tcgtcggaag gtcgggaggt caggccggtt cggggttctg
tggtcgacga tccgcgaaag 1860 gtaggttcaa ccgaccttgg aggtcgggga
cccgatcgag tctatcagag acgtcggcgt 1920 cccctgagac aaggggtgcg
tacagagact ggggatgtct ccgaaccttc cggtttttct 1980 tcgtccttaa
cgtggttcga ttgtcgaggg gtgacgaggc cccgtcggtg gggtaccttt 2040
agacccggac cctcaaccct tcgtctcgga ggttcttgga aagagtttcg ggtcctcttc
2100 gcggggctcg gcaccatagg accgcacgac accctggtgt tgaagtggcg
ttgaggtcac 2160 tcgaccgtag agcaggtgag ggaggttgtg gggaaagaga
agcacctcga aggtcagtac 2220 tgggtgtctc ggtcacacac ctcttcgagg
ttcgagggag gagactgggt gacggtcgtc 2280 ggggagagag gcaggagttg
agaaggtctg gaaggtcggg ggtccggaga aaggagagga 2340 caggatcggg
taggaggtcg ttggacaggt cgaggagcga cagtaggaat ctcctcctcc 2400
tcctagtcct gtcgcacgag tgggggctcc tccatcggga cacagacctc gagtcactac
2460 ccctcctctg tgggtgcttg tcacatagag gatacggttc tcgaggaagg
ggcggttgtt 2520 ggataccgat atagtcgtat ggttggacga gtcctgaccg
tctgtacccg tctcgaccgc 2580 ccccgcaccc cagactccaa cccttgaatg
acataggtgg agccgggacg gggtggggat 2640 gtgggtcgct cccgagggac
cggttaccaa ccccgagtcg aagactcctg ttacaggggt 2700 cgcggtcccg
gtcggaccaa tcgagaagac taccgaggaa ggagcgacta tgagtgaaac 2760
gagcacggga ccgtcaccga cacctatcga aaccggagtc agacctaggg tcccttcgac
2820 tgacacagaa gtgactacgg agtagtggag ggaggggagc cccactagag
agggactggg 2880 cttcgaagag agacggaaac accctcacct ccggtctgac
caaccttcta cgactctagt 2940 cggtgtgggt ctccgacccc tcccccgacg
gagggaccgg aggactaaga tcccggagaa 3000 gggtcgcttc aaccgattga
ccacgacacg ggttccgacc actaaggagg acttaacagg 3060 gactcttccg
gtcttctcgt gggtctggtg agaggacaga caggggaccg aaagagtgta 3120
cacctccaga accggatacg aagagacatt atcttcaggt ggcagtgatc cgaagacctc
3180 tcgagacagt aaccctaaca attttattta ctttcgtttg gttttatact
agtgccctca 3240 gaacctaagg gtgactcttg ttctgtcgta gaagtcctgt
cgtctgagag gtgttggtct 3300 tggaaaccgg attcattcgg accgaggcct
cgagggtgga ttcacctagt acctttcttc 3360 ccttcggttg gtccagaagt
ccttcctgtc tttacaaaaa accactcccg ataccacctc 3420 ctggacacct
tctcgggaga gtatagatga acctgaggag ggaatctccg gtcgagttgg 3480
gaaaggggtc agtgtggtac gttcctttga tttcctcttt ccagcaccta cgtcacccgg
3540 gatatgtcgc agtgtcagtt acgaagtttc actctagtta cctcctctga
cttcctttcc 3600 tgcgtccctt tgtcccttgg ttacgcgata agagtaagat
ggcggtgaga ctcgaattcc 3660 ttgaattaag atattttgac atttctgc 3688 40
1015 PRT Mus musculus 40 Met Gly Gln Gly Glu Glu Pro Arg Ala Ala
Met Gly Ser Gly Gly Thr 1 5 10 15 Gly Leu Leu Gly Thr Glu Trp Pro
Leu Pro Leu Leu Leu Leu Phe Ile 20 25 30 Met Gly Gly Glu Ala Leu
Asp Ser Pro Pro Gln Ile Leu Val His Pro 35 40 45 Gln Asp Gln Leu
Leu Gln Gly Ser Gly Pro Ala Lys Met Arg Cys Arg 50 55 60 Ser Ser
Gly Gln Pro Pro Pro Thr Ile Arg Trp Leu Leu Asn Gly Gln 65 70 75 80
Pro Leu Ser Met Ala Thr Pro Asp Leu His Tyr Leu Leu Pro Asp Gly 85
90 95 Thr Leu Leu Leu His Arg Pro Ser Val Gln Gly Arg Pro Gln Asp
Asp 100 105 110 Gln Asn Ile Leu Ser Ala Ile Leu Gly Val Tyr Thr Cys
Glu Ala Ser 115 120 125 Asn Arg Leu Gly Thr Ala Val Ser Arg Gly Ala
Arg Leu Ser Val Ala 130 135 140 Val Leu Gln Glu Asp Phe Gln Ile Gln
Pro Arg Asp Thr Val Ala Val 145 150 155 160 Val Gly Glu Ser Leu Val
Leu Glu Cys Gly Pro Pro Trp Gly Tyr Pro 165 170 175 Lys Pro Ser Val
Ser Trp Trp Lys Asp Gly Lys Pro Leu Val Leu Gln 180 185 190 Pro Gly
Arg Arg Thr Val Ser Gly Asp Ser Leu Met Val Ser Arg Ala 195 200 205
Glu Lys Asn Asp Ser Gly Thr Tyr Met Cys Met Ala Thr Asn Asn Ala 210
215 220 Gly Gln Arg Glu Ser Arg Ala Ala Arg Val Ser Ile Gln Glu Ser
Gln 225 230 235 240 Asp His Lys Glu His Leu Glu Leu Leu Ala Val Arg
Ile Gln Leu Glu 245 250 255 Asn Val Thr Leu Leu Asn Pro Glu Pro Val
Lys Gly Pro Lys Pro Gly 260 265 270 Pro Ser Val Trp Leu Ser Trp Lys
Val Ser Gly Pro Ala Ala Pro Ala 275 280 285 Glu Ser Tyr Thr Ala Leu
Phe Arg Thr Gln Arg Ser Pro Arg Asp Gln 290 295 300 Gly Ser Pro Trp
Thr Glu Val Leu Leu Arg Gly Leu Gln Ser Ala Lys 305 310 315 320 Leu
Gly Gly Leu His Trp Gly Gln Asp Tyr Glu Phe Lys Val Arg Pro 325 330
335 Ser Ser Gly Arg Ala Arg Gly Pro Asp Ser Asn Val Leu Leu Leu Arg
340 345 350 Leu Pro Glu Gln Val Pro Ser Ala Pro Pro Gln Gly Val Thr
Leu Arg 355 360 365 Ser Gly Asn Gly Ser Val Phe Val Ser Trp Ala Pro
Pro Pro Ala Glu 370 375 380 Ser His Asn Gly Val Ile Arg Gly Tyr Gln
Val Trp Ser Leu Gly Asn 385 390 395 400 Ala Ser Leu Pro Ala Ala Asn
Trp Thr Val Val Gly Glu Gln Thr Gln 405 410 415 Leu Glu Ile Ala Thr
Arg Leu Pro Gly Ser Tyr Cys Val Gln Val Ala 420 425 430 Ala Val Thr
Gly Ala Gly Ala Gly Glu Leu Ser Thr Pro Val Cys Leu 435 440 445 Leu
Leu Glu Gln Ala Met Glu Gln Ser Ala Arg Asp Pro Arg Lys His 450 455
460 Val Pro Trp Thr Leu Glu Gln Leu Arg Ala Thr Leu Arg Arg Pro Glu
465 470 475 480 Val Ile Ala Ser Ser Ala Val Leu Leu Trp Leu Leu Leu
Leu Gly Ile 485 490 495 Thr Val Cys Ile Tyr Arg Arg Arg Lys Ala Gly
Val His Leu Gly Pro 500 505 510 Gly Leu Tyr Arg Tyr Thr Ser Glu Asp
Ala Ile Leu Lys His Arg Met 515 520 525 Asp His Ser Asp Ser Pro Trp
Leu Ala Asp Thr Trp Arg Ser Thr Ser 530 535 540 Gly Ser Arg Asp Leu
Ser Ser Ser Ser Ser Leu Ser Ser Arg Leu Gly 545 550 555 560 Leu Asp
Pro Arg Asp Pro Leu Glu Gly Arg Arg Ser Leu Ile Ser Trp 565 570 575
Asp Pro Arg Ser Pro Gly Val Pro Leu Leu Pro Asp Thr Ser Thr Phe 580
585 590 Tyr Gly Ser Leu Ile Ala Glu Gln Pro Ser Ser Pro Pro Val Arg
Pro 595 600 605 Ser Pro Lys Thr Pro Ala Ala Arg Arg Phe Pro Ser Lys
Leu Ala Gly 610 615 620 Thr Ser Ser Pro Trp Ala Ser Ser Asp Ser Leu
Cys Ser Arg Arg Gly 625 630 635 640 Leu Cys Ser Pro Arg Met Ser Leu
Thr Pro Thr Glu Ala Trp Lys Ala 645 650 655 Lys Lys Lys Gln Glu Leu
His Gln Ala Asn Ser Ser Pro Leu Leu Arg 660 665 670 Gly Ser His Pro
Met Glu Ile Trp Ala Trp Glu Leu Gly Ser Arg Ala 675 680 685 Ser Lys
Asn Leu Ser Gln Ser Pro Gly Glu Ala Pro Arg Ala Val Val 690 695 700
Ser Trp Arg Ala Val Gly Pro Gln Leu His Arg Asn Ser Ser Glu Leu 705
710 715 720 Ala Ser Arg Pro Leu Pro Pro Thr Pro Leu Ser Leu Arg Gly
Ala Ser 725 730 735 Ser His Asp Pro Gln Ser Gln Cys Val Glu Lys Leu
Gln Ala Pro Ser 740 745 750 Ser Asp Pro Leu Pro Ala Ala Pro Leu Ser
Val Leu Asn Ser Ser Arg 755 760 765 Pro Ser Ser Pro Gln Ala Ser Phe
Leu Ser Cys Pro Ser Pro Ser Ser 770 775 780 Ser Asn Leu Ser Ser Ser
Ser Leu Ser Ser Leu Glu Glu Glu Glu Asp 785 790 795 800 Gln Asp Ser
Val Leu Thr Pro Glu Glu Val Ala Leu Cys Leu Glu Leu 805 810 815 Ser
Asp Gly Glu Glu Thr Pro Thr Asn Ser Val Ser Pro Met Pro Arg 820 825
830 Ala Pro Ser Pro Pro Thr Thr Tyr Gly Tyr Ile Ser Ile Pro Thr Cys
835 840 845 Ser Gly Leu Ala Asp Met Gly Arg Ala Gly Gly Gly Val Gly
Ser Glu 850 855 860 Val Gly Asn Leu Leu Tyr Pro Pro Arg Pro Cys Pro
Thr Pro Thr Pro 865 870 875 880 Ser Glu Gly Ser Leu Ala Asn Gly Trp
Gly Ser Ala Ser Glu Asp Asn 885 890 895 Val Pro Ser Ala Arg Ala Ser
Leu Val Ser Ser Ser Asp Gly Ser Phe 900 905 910 Leu Ala Asp Thr His
Phe Ala Arg Ala Leu Ala Val Ala Val Asp Ser 915 920 925 Phe Gly Leu
Ser Leu Asp Pro Arg Glu Ala Asp Cys Val Phe Thr Asp 930 935 940 Ala
Ser Ser Pro Pro Ser Pro Arg Gly Asp Leu Ser Leu Thr Arg Ser 945 950
955 960 Phe Ser Leu Pro Leu Trp Glu Trp Arg Pro Asp Trp Leu Glu Asp
Ala 965 970 975 Glu Ile Ser His Thr Gln Arg Leu Gly Arg Gly Leu Pro
Pro Trp Pro 980 985 990 Pro Asp Ser Arg Ala Ser Ser Gln Arg Ser Trp
Leu Thr Gly Ala Val 995 1000 1005 Pro Lys Ala Gly Asp Ser Ser 1010
1015 41 39 PRT Mus musculus 41 Met Phe Phe Gly Glu Gly Tyr Gly Gly
Gly Pro Val Glu Glu Pro Ser 1 5 10 15 His Ile Tyr Leu Asp Ser Ser
Leu Arg Gly Gln Leu Asn Pro Phe Pro 20 25 30 Ser His Thr Met Gln
Gly Asn 35 42 34 PRT Mus musculus 42 Met Glu Glu Thr Glu Gly Lys
Asp Ala Gly Lys Gln Gly Thr Asn Ala 1 5 10 15 Leu Phe Ser Phe Tyr
Arg His Ser Glu Leu Lys Glu Leu Asn Ser Ile 20 25 30 Lys Leu 43
1075 PRT Homo sapiens 43 Met Gly Ser Gly Gly Asp Ser Leu Leu Gly
Gly Arg Gly Ser Leu Pro 1 5 10 15 Leu Leu Leu Leu Leu Ile Met Gly
Gly Met Ala Gln Asp Ser Pro Pro 20 25 30 Gln Ile Leu Val His Pro
Gln Asp Gln Leu Phe Gln Gly Pro Gly Pro 35 40 45 Ala Arg Met Ser
Cys Gln Ala Ser Gly Gln Pro Pro Pro Thr Ile Arg 50 55 60 Trp Leu
Leu Asn Gly Gln Pro Leu Ser Met Val Pro Pro Asp Pro His 65 70 75 80
His Leu Leu Pro Asp Gly Thr Leu Leu Leu Leu Gln Pro Pro Ala Arg 85
90 95 Gly His Ala His Asp Gly Gln Ala Leu Ser Thr Asp Leu Gly Val
Tyr 100 105 110 Thr Cys Glu Ala Ser Asn Arg Leu Gly Thr Ala Val Ser
Arg Gly Ala 115 120 125 Arg Leu Ser Val Ala Val Leu Arg Glu Asp Phe
Gln Ile Gln Pro Arg 130 135 140 Asp Met Val Ala Val Val Gly Glu Gln
Phe Thr Leu Glu Cys Gly Pro 145 150 155 160 Pro Trp Gly His Pro Glu
Pro Thr Val Ser Trp Trp Lys Asp Gly Lys 165 170 175 Pro Leu Ala Leu
Gln Pro Gly Arg His Thr Val Ser Gly Gly Ser Leu 180 185 190 Leu Met
Ala Arg Ala Glu Lys Ser Asp Glu Gly Thr Tyr Met Cys Val 195 200 205
Ala Thr Asn Ser Ala Gly His Arg Glu Ser Arg Ala Ala Arg Val Ser 210
215 220 Ile Gln Glu Pro Gln Asp Tyr Thr Glu Pro Val Glu Leu Leu Ala
Val 225 230 235 240 Arg Ile Gln Leu Glu Asn Val Thr Leu Leu Asn Pro
Asp Pro Ala Glu 245 250 255 Gly Pro Lys Pro Arg Pro Ala Val Trp Leu
Ser Trp Lys Val Ser Gly 260 265 270 Pro Ala Ala Pro Ala Gln Ser Tyr
Thr Ala Leu Phe Arg Thr Gln Thr 275 280 285 Ala Pro Gly Gly Gln Gly
Ala Pro Trp Ala Glu Glu Leu Leu Ala Gly 290 295 300 Trp Gln Ser Ala
Glu Leu Gly Gly Leu His Trp Gly Gln Asp Tyr Glu 305 310 315 320 Phe
Lys Val Arg Pro Ser Ser Gly Arg Ala Arg Gly Pro Asp Ser Asn 325 330
335 Val Leu Leu Leu Arg Leu Pro Glu Lys Val Pro Ser Ala Pro Pro Gln
340 345 350 Glu Val Thr Leu Lys Pro Gly Asn Gly Thr Val Phe Val Ser
Trp Val 355 360 365 Pro Pro Pro Ala Glu Asn His Asn Gly Ile Ile Arg
Gly Tyr Gln Val 370 375 380 Trp Ser Leu Gly Asn Thr Ser Leu Pro Pro
Ala Asn Trp Thr Val Val 385 390 395 400 Gly Glu Gln Thr Gln Leu Glu
Ile Ala Thr His Met Pro Gly Ser Tyr 405 410 415 Cys Val Gln Val Ala
Ala Val Thr Gly Ala Gly Ala Gly Glu Pro Ser 420 425 430 Arg Pro Val
Cys Leu Leu Leu Glu Gln Ala Met Glu Arg Ala Thr Gln 435 440 445 Glu
Pro Ser Glu His Gly Pro Trp Thr Leu Glu Gln Leu Arg Ala Thr 450 455
460 Leu Lys Arg Pro Glu Val Ile Ala Thr Cys Gly Val Ala Leu Trp Leu
465 470 475 480 Leu Leu Leu Gly Thr Ala Val Cys Ile His Arg Arg Arg
Arg Ala Arg 485 490 495 Val His Leu Gly Pro Gly Leu Tyr Arg Tyr Thr
Ser Glu Asp Ala Ile 500 505 510 Leu Lys His Arg Met Asp His Ser Asp
Ser Gln Trp Leu Ala Asp Thr 515 520 525 Trp Arg Ser Thr Ser Gly Ser
Arg Asp Leu Ser Ser Ser Ser Ser Leu 530 535 540 Ser Ser Arg Leu Gly
Ala Asp Ala Arg Asp Pro Leu Asp Cys Arg Arg 545 550 555 560 Ser Leu
Leu Ser Trp Asp Ser Arg Ser Pro Gly Val Pro Leu Leu Pro 565 570 575
Asp Thr Ser Thr Phe Tyr Gly Ser Leu Ile Ala Glu Leu Pro Ser Ser 580
585 590 Thr Pro Ala Arg Pro Ser Pro Gln Val Pro Ala Val Arg Arg Leu
Pro 595 600 605 Pro Gln Leu Ala Gln Leu Ser Ser Pro Cys Ser Ser Ser
Asp Ser Leu 610 615 620 Cys Ser Arg Arg Gly Leu Ser Ser Pro Arg Leu
Ser Leu Ala Pro Ala 625 630 635 640 Glu Ala Trp Lys Ala Lys Lys Lys
Gln Glu Leu Gln His Ala Asn Ser 645 650 655 Ser Pro Leu Leu Arg Gly
Ser His Ser Leu Glu Leu Arg Ala Cys Glu
660 665 670 Leu Gly Asn Arg Gly Ser Lys Asn Leu Ser Gln Ser Pro Gly
Ala Val 675 680 685 Pro Gln Ala Leu Val Ala Trp Arg Ala Leu Gly Pro
Lys Leu Leu Ser 690 695 700 Ser Ser Asn Glu Leu Val Thr Arg His Leu
Pro Pro Ala Pro Leu Phe 705 710 715 720 Pro His Glu Thr Pro Pro Thr
Gln Ser Gln Gln Thr Gln Pro Pro Val 725 730 735 Ala Pro Gln Ala Pro
Ser Ser Ile Leu Leu Pro Ala Ala Pro Ile Pro 740 745 750 Ile Leu Ser
Pro Cys Ser Pro Pro Ser Pro Gln Ala Ser Ser Leu Ser 755 760 765 Gly
Pro Ser Pro Ala Ser Ser Arg Leu Ser Ser Ser Ser Leu Ser Ser 770 775
780 Leu Gly Glu Asp Gln Asp Ser Val Leu Thr Pro Glu Glu Val Ala Leu
785 790 795 800 Cys Leu Glu Leu Ser Glu Gly Glu Glu Thr Pro Arg Asn
Ser Val Ser 805 810 815 Pro Met Pro Arg Ala Pro Ser Pro Pro Thr Thr
Tyr Gly Tyr Ile Ser 820 825 830 Val Pro Thr Ala Ser Glu Phe Thr Asp
Met Gly Arg Thr Gly Gly Gly 835 840 845 Val Gly Pro Lys Gly Gly Val
Leu Leu Cys Pro Pro Arg Pro Cys Leu 850 855 860 Thr Pro Thr Pro Ser
Glu Gly Ser Leu Ala Asn Gly Trp Gly Ser Ala 865 870 875 880 Ser Glu
Asp Asn Ala Ala Ser Ala Arg Ala Ser Leu Val Ser Ser Ser 885 890 895
Asp Gly Ser Phe Leu Ala Asp Ala His Phe Ala Arg Ala Leu Ala Val 900
905 910 Ala Val Asp Ser Phe Gly Phe Gly Leu Glu Pro Arg Glu Ala Asp
Cys 915 920 925 Val Phe Ile Asp Ala Ser Ser Pro Pro Ser Pro Arg Asp
Glu Ile Phe 930 935 940 Leu Thr Pro Asn Leu Ser Leu Pro Leu Trp Glu
Trp Arg Pro Asp Trp 945 950 955 960 Leu Glu Asp Met Glu Val Ser His
Thr Gln Arg Leu Gly Arg Gly Met 965 970 975 Pro Pro Trp Pro Pro Glu
Leu Ser Asp Leu Phe Pro Glu Lys Ser Ala 980 985 990 Pro Leu Ser Tyr
Ala Gln Gly Trp Cys Phe Ser Cys Arg Leu Leu Leu 995 1000 1005 Asn
Arg Val Pro Glu Thr Ser Gln Thr Gly Ile Arg Thr Thr Ser Pro 1010
1015 1020 Val Pro Pro Thr Arg Pro Gly Leu Trp Cys Val Gly Leu Gly
Leu Cys 1025 1030 1035 1040 Phe Ser Ala Ala Gly Val His Leu Pro Lys
Pro Pro Glu Ser Ser Pro 1045 1050 1055 Ser Thr Ile Val Lys Thr Asn
Glu Asn Lys Ile Arg Ala Lys Leu Thr 1060 1065 1070 Trp Ser Pro 1075
44 38 PRT Mus musculus 44 Ile Val Pro Glu Lys Ala Arg Arg Ala Pro
Arg Pro Leu Ser Cys Leu 1 5 10 15 Ser Pro Gly Phe Leu Thr Cys Gly
Gly Leu Gly Leu Cys Phe Ser Val 20 25 30 Ile Glu Val His Arg His 35
45 10 PRT Mus musculus 45 Ala Ser Gly Glu Leu Cys His Trp Asp Cys 1
5 10 46 5 PRT Mus musculus 46 Lys Gln Thr Lys Ile 1 5 47 7 PRT Mus
musculus 47 Ser Arg Glu Ser Trp Ile Pro 1 5 48 1005 PRT Mus
musculus 48 Met Gly Ser Gly Gly Thr Gly Leu Leu Gly Thr Glu Trp Pro
Leu Pro 1 5 10 15 Leu Leu Leu Leu Phe Ile Met Gly Gly Glu Ala Leu
Asp Ser Pro Pro 20 25 30 Gln Ile Leu Val His Pro Gln Asp Gln Leu
Leu Gln Gly Ser Gly Pro 35 40 45 Ala Lys Met Arg Cys Arg Ser Ser
Gly Gln Pro Pro Pro Thr Ile Arg 50 55 60 Trp Leu Leu Asn Gly Gln
Pro Leu Ser Met Ala Thr Pro Asp Leu His 65 70 75 80 Tyr Leu Leu Pro
Asp Gly Thr Leu Leu Leu His Arg Pro Ser Val Gln 85 90 95 Gly Arg
Pro Gln Asp Asp Gln Asn Ile Leu Ser Ala Ile Leu Gly Val 100 105 110
Tyr Thr Cys Glu Ala Ser Asn Arg Leu Gly Thr Ala Val Ser Arg Gly 115
120 125 Ala Arg Leu Ser Val Ala Val Leu Gln Glu Asp Phe Gln Ile Gln
Pro 130 135 140 Arg Asp Thr Val Ala Val Val Gly Glu Ser Leu Val Leu
Glu Cys Gly 145 150 155 160 Pro Pro Trp Gly Tyr Pro Lys Pro Ser Val
Ser Trp Trp Lys Asp Gly 165 170 175 Lys Pro Leu Val Leu Gln Pro Gly
Arg Arg Thr Val Ser Gly Asp Ser 180 185 190 Leu Met Val Ser Arg Ala
Glu Lys Asn Asp Ser Gly Thr Tyr Met Cys 195 200 205 Met Ala Thr Asn
Asn Ala Gly Gln Arg Glu Ser Arg Ala Ala Arg Val 210 215 220 Ser Ile
Gln Glu Ser Gln Asp His Lys Glu His Leu Glu Leu Leu Ala 225 230 235
240 Val Arg Ile Gln Leu Glu Asn Val Thr Leu Leu Asn Pro Glu Pro Val
245 250 255 Lys Gly Pro Lys Pro Gly Pro Ser Val Trp Leu Ser Trp Lys
Val Ser 260 265 270 Gly Pro Ala Ala Pro Ala Glu Ser Tyr Thr Ala Leu
Phe Arg Thr Gln 275 280 285 Arg Ser Pro Arg Asp Gln Gly Ser Pro Trp
Thr Glu Val Leu Leu Arg 290 295 300 Gly Leu Gln Ser Ala Lys Leu Gly
Gly Leu His Trp Gly Gln Asp Tyr 305 310 315 320 Glu Phe Lys Val Arg
Pro Ser Ser Gly Arg Ala Arg Gly Pro Asp Ser 325 330 335 Asn Val Leu
Leu Leu Arg Leu Pro Glu Gln Val Pro Ser Ala Pro Pro 340 345 350 Gln
Gly Val Thr Leu Arg Ser Gly Asn Gly Ser Val Phe Val Ser Trp 355 360
365 Ala Pro Pro Pro Ala Glu Ser His Asn Gly Val Ile Arg Gly Tyr Gln
370 375 380 Val Trp Ser Leu Gly Asn Ala Ser Leu Pro Ala Ala Asn Trp
Thr Val 385 390 395 400 Val Gly Glu Gln Thr Gln Leu Glu Ile Ala Thr
Arg Leu Pro Gly Ser 405 410 415 Tyr Cys Val Gln Val Ala Ala Val Thr
Gly Ala Gly Ala Gly Glu Leu 420 425 430 Ser Thr Pro Val Cys Leu Leu
Leu Glu Gln Ala Met Glu Gln Ser Ala 435 440 445 Arg Asp Pro Arg Lys
His Val Pro Trp Thr Leu Glu Gln Leu Arg Ala 450 455 460 Thr Leu Arg
Arg Pro Glu Val Ile Ala Ser Ser Ala Val Leu Leu Trp 465 470 475 480
Leu Leu Leu Leu Gly Ile Thr Val Cys Ile Tyr Arg Arg Arg Lys Ala 485
490 495 Gly Val His Leu Gly Pro Gly Leu Tyr Arg Tyr Thr Ser Glu Asp
Ala 500 505 510 Ile Leu Lys His Arg Met Asp His Ser Asp Ser Pro Trp
Leu Ala Asp 515 520 525 Thr Trp Arg Ser Thr Ser Gly Ser Arg Asp Leu
Ser Ser Ser Ser Ser 530 535 540 Leu Ser Ser Arg Leu Gly Leu Asp Pro
Arg Asp Pro Leu Glu Gly Arg 545 550 555 560 Arg Ser Leu Ile Ser Trp
Asp Pro Arg Ser Pro Gly Val Pro Leu Leu 565 570 575 Pro Asp Thr Ser
Thr Phe Tyr Gly Ser Leu Ile Ala Glu Gln Pro Ser 580 585 590 Ser Pro
Pro Val Arg Pro Ser Pro Lys Thr Pro Ala Ala Arg Arg Phe 595 600 605
Pro Ser Lys Leu Ala Gly Thr Ser Ser Pro Trp Ala Ser Ser Asp Ser 610
615 620 Leu Cys Ser Arg Arg Gly Leu Cys Ser Pro Arg Met Ser Leu Thr
Pro 625 630 635 640 Thr Glu Ala Trp Lys Ala Lys Lys Lys Gln Glu Leu
His Gln Ala Asn 645 650 655 Ser Ser Pro Leu Leu Arg Gly Ser His Pro
Met Glu Ile Trp Ala Trp 660 665 670 Glu Leu Gly Ser Arg Ala Ser Lys
Asn Leu Ser Gln Ser Pro Gly Glu 675 680 685 Ala Pro Arg Ala Val Val
Ser Trp Arg Ala Val Gly Pro Gln Leu His 690 695 700 Arg Asn Ser Ser
Glu Leu Ala Ser Arg Pro Leu Pro Pro Thr Pro Leu 705 710 715 720 Ser
Leu Arg Gly Ala Ser Ser His Asp Pro Gln Ser Gln Cys Val Glu 725 730
735 Lys Leu Gln Ala Pro Ser Ser Asp Pro Leu Pro Ala Ala Pro Leu Ser
740 745 750 Val Leu Asn Ser Ser Arg Pro Ser Ser Pro Gln Ala Ser Phe
Leu Ser 755 760 765 Cys Pro Ser Pro Ser Ser Ser Asn Leu Ser Ser Ser
Ser Leu Ser Ser 770 775 780 Leu Glu Glu Glu Glu Asp Gln Asp Ser Val
Leu Thr Pro Glu Glu Val 785 790 795 800 Ala Leu Cys Leu Glu Leu Ser
Asp Gly Glu Glu Thr Pro Thr Asn Ser 805 810 815 Val Ser Pro Met Pro
Arg Ala Pro Ser Pro Pro Thr Thr Tyr Gly Tyr 820 825 830 Ile Ser Ile
Pro Thr Cys Ser Gly Leu Ala Asp Met Gly Arg Ala Gly 835 840 845 Gly
Gly Val Gly Ser Glu Val Gly Asn Leu Leu Tyr Pro Pro Arg Pro 850 855
860 Cys Pro Thr Pro Thr Pro Ser Glu Gly Ser Leu Ala Asn Gly Trp Gly
865 870 875 880 Ser Ala Ser Glu Asp Asn Val Pro Ser Ala Arg Ala Ser
Leu Val Ser 885 890 895 Ser Ser Asp Gly Ser Phe Leu Ala Asp Thr His
Phe Ala Arg Ala Leu 900 905 910 Ala Val Ala Val Asp Ser Phe Gly Leu
Ser Leu Asp Pro Arg Glu Ala 915 920 925 Asp Cys Val Phe Thr Asp Ala
Ser Ser Pro Pro Ser Pro Arg Gly Asp 930 935 940 Leu Ser Leu Thr Arg
Ser Phe Ser Leu Pro Leu Trp Glu Trp Arg Pro 945 950 955 960 Asp Trp
Leu Glu Asp Ala Glu Ile Ser His Thr Gln Arg Leu Gly Arg 965 970 975
Gly Leu Pro Pro Trp Pro Pro Asp Ser Arg Ala Ser Ser Gln Arg Ser 980
985 990 Trp Leu Thr Gly Ala Val Pro Lys Ala Gly Asp Ser Ser 995
1000 1005 49 3651 DNA Homo sapiens 49 agtgctcggg acaaggacat
agggctgaga gtagccatgg gctctggagg agacagcctc 60 ctggggggca
ggggttccct gcctctgctg ctcctgctca tcatgggagg catggctcag 120
gactccccgc cccagatcct agtccacccc caggaccagc tgttccaggg ccctggccct
180 gccaggatga gctgccaagc ctcaggccag ccacctccca ccatccgctg
gttgctgaat 240 gggcagcccc tgagcatggt gcccccagac ccacaccacc
tcctgcctga tgggaccctt 300 ctgctgctac agccccctgc ccggggacat
gcccacgatg gccaggccct gtccacagac 360 ctgggtgtct acacatgtga
ggccagcaac cggcttggca cggcagtcag cagaggcgct 420 cggctgtctg
tggctgtcct ccgggaggat ttccagatcc agcctcggga catggtggct 480
gtggtgggtg agcagtttac tctggaatgt gggccgccct ggggccaccc agagcccaca
540 gtctcatggt ggaaagatgg gaaacccctg gccctccagc ccggaaggca
cacagtgtcc 600 ggggggtccc tgctgatggc aagagcagag aagagtgacg
aagggaccta catgtgtgtg 660 gccaccaaca gcgcaggaca tagggagagc
cgcgcagccc gggtttccat ccaggagccc 720 caggactaca cggagcctgt
ggagcttctg gctgtgcgaa ttcagctgga aaatgtgaca 780 ctgctgaacc
cggatcctgc agagggcccc aagcctagac cggcggtgtg gctcagctgg 840
aaggtcagtg gccctgctgc gcctgcccaa tcttacacgg ccttgttcag gacccagact
900 gccccgggag gccagggagc tccgtgggca gaggagctgc tggccggctg
gcagagcgca 960 gagcttggag gcctccactg gggccaagac tacgagttca
aagtgagacc atcctctggc 1020 cgggctcgag gccctgacag caacgtgctg
ctcctgaggc tgccggaaaa agtgcccagt 1080 gccccacctc aggaagtgac
tctaaagcct ggcaatggca ctgtctttgt gagctgggtc 1140 ccaccacctg
ctgaaaacca caatggcatc atccgtggct accaggtctg gagcctgggc 1200
aacacatcac tgccaccagc caactggact gtagttggtg agcagaccca gctggaaatc
1260 gccacccata tgccaggctc ctactgcgtg caagtggctg cagtcactgg
tgctggagct 1320 ggggagccca gtagacctgt ctgcctcctt ttagagcagg
ccatggagcg agccacccaa 1380 gaacccagtg agcatggtcc ctggaccctg
gagcagctga gggctacctt gaagcggcct 1440 gaggtcattg ccacctgcgg
tgttgcactc tggctgctgc ttctgggcac cgccgtgtgt 1500 atccaccgcc
ggcgccgagc tagggtgcac ctgggcccag gtctgtacag atataccagt 1560
gaggatgcca tcctaaaaca caggatggat cacagtgact cccagtggtt ggcagacact
1620 tggcgttcca cctctggctc tcgggacctg agcagcagca gcagcctcag
cagtcggctg 1680 ggggcggatg cccgggaccc actagactgt cgtcgctcct
tgctctcctg ggactcccga 1740 agccccggcg tgcccctgct tccagacacc
agcacttttt atggctccct catcgctgag 1800 ctgccctcca gtaccccagc
caggccaagt ccccaggtcc cagctgtcag gcgcctccca 1860 ccccagctgg
cccagctctc cagcccctgt tccagctcag acagcctctg cagccgcagg 1920
ggactctctt ctccccgctt gtctctggcc cctgcagagg cttggaaggc caaaaagaag
1980 caggagctgc agcatgccaa cagttcccca ctgctccggg gcagccactc
cttggagctc 2040 cgggcctgtg agttaggaaa tagaggttcc aagaaccttt
cccaaagccc aggagctgtg 2100 ccccaagctc tggttgcctg gcgggccctg
ggaccgaaac tcctcagctc ctcaaatgag 2160 ctggttactc gtcatctccc
tccagcaccc ctctttcctc atgaaactcc cccaactcag 2220 agtcaacaga
cccagcctcc ggtggcacca caggctccct cctccatcct gctgccagca 2280
gcccccatcc ccatccttag cccctgcagt ccccctagcc cccaggcctc ttccctctct
2340 ggccccagcc cagcttccag tcgcctgtcc agctcctcac tgtcatccct
gggggaggat 2400 caagacagcg tgctgacccc tgaggaggta gccctgtgct
tggaactcag tgagggtgag 2460 gagactccca ggaacagcgt ctctcccatg
ccaagggctc cttcaccccc caccacctat 2520 gggtacatca gcgtcccaac
agcctcagag ttcacggaca tgggcaggac tggaggaggg 2580 gtggggccca
aggggggagt cttgctgtgc ccacctcggc cctgcctcac ccccaccccc 2640
agcgagggct ccttagccaa tggttggggc tcagcctctg aggacaatgc cgccagcgcc
2700 agagccagcc ttgtcagctc ctccgatggc tccttcctcg ctgatgctca
ctttgcccgg 2760 gccctggcag tggctgtgga tagctttggt ttcggtctag
agcccaggga ggcagactgc 2820 gtcttcatag atgcctcatc acctccctcc
ccacgggatg agatcttcct gacccccaac 2880 ctctccctgc ccctgtggga
gtggaggcca gactggttgg aagacatgga ggtcagccac 2940 acccagcggc
tgggaagggg gatgcctccc tggccccctg aactctcaga tctcttccca 3000
gagaagtcag ctccactgtc gtatgcccaa ggctggtgct tctcctgtag attactcctg
3060 aaccgtgtcc ctgagacttc ccagacggga atcagaacca cttctcctgt
tccacccaca 3120 agacctgggc tgtggtgtgt gggtcttggc ctgtgtttct
ctgcagctgg ggtccacctt 3180 cccaagcctc cagagagttc tccctccacg
attgtgaaaa caaatgaaaa caaaattaga 3240 gcaaagctga cctggagccc
tcagggagca aaacatcatc tccacctgac tcctagccac 3300 tgctttctcc
tctgtgccat ccactcccac caccaggttg ttttggcctg aggagcagcc 3360
ctgcctgctg ctcttccccc accatttgga tcacaggaag tggaggagcc agaggtgcct
3420 ttgtggagga cagcagtggc tgctgggaga gggctgtgga ggaaggagct
tctcggagcc 3480 ccctctcagc cttacctggg cccctcctct agagaagagc
tcaactctct cccaacctca 3540 ccatggaaag aaaataatta tgaatgccac
tgaggcactg aggccctacc tcatgccaaa 3600 caaagggttc aaggctgggt
ctagcgagga tgctgaagga agggaggtat g 3651 50 3607 DNA Mus musculus 50
agtgtatggg acaaggagag gagccgagag cagccatggg ctctggagga acgggcctcc
60 tggggacgga gtggcctctg cctctgctgc tgcttttcat catgggaggt
gaggctctgg 120 attctccacc ccagatccta gttcaccccc aggaccagct
acttcagggc tctggcccag 180 ccaagatgag gtgcagatca tccggccaac
cacctcccac tatccgctgg ctgctgaatg 240 ggcagcccct cagcatggcc
accccagacc tacattacct tttgccggat gggaccctcc 300 tgttacatcg
gccctctgtc cagggacggc cacaagatga ccagaacatc ctctcagcaa 360
tcctgggtgt ctacacatgt gaggccagca accggctggg cacagcagtg agccggggtg
420 ctaggctgtc tgtggctgtc ctccaggagg acttccagat ccaacctcgg
gacacagtgg 480 ccgtggtggg agagagcttg gttcttgagt gtggtcctcc
ctggggctac ccaaaaccct 540 cggtctcatg gtggaaagac gggaaacccc
tggtcctcca gccagggagg cgcacagtat 600 ctggggattc cctgatggtg
tcaagagcag agaagaatga ctcggggacc tatatgtgta 660 tggccaccaa
caatgctggg caacgggaga gccgagcagc cagggtgtct atccaggaat 720
cccaggacca caaggaacat ctagagcttc tggctgttcg cattcagctg gaaaatgtga
780 ccctgctaaa ccccgaacct gtaaaaggtc ccaagcctgg gccatccgtg
tggctcagct 840 ggaaggtgag cggccctgct gcacctgctg agtcatacac
agctctgttc aggactcaga 900 ggtcccccag ggaccaagga tctccatgga
cagaggtgct gctgcgtggc ttgcagagtg 960 caaagcttgg gggtctccac
tggggccaag actatgaatt caaagtgaga ccgtcctccg 1020 gccgggctcg
aggccctgac agcaatgtgt tgctcctgag gctgcctgaa caggtgccca 1080
gtgccccacc tcaaggagtg accttaagat ctggcaacgg tagtgtcttt gtgagttggg
1140 ctccaccacc tgctgaaagc cataatggtg tcatccgtgg ttaccaggtc
tggagcctgg 1200 gcaatgcctc attgcctgct gccaactgga ccgtagtggg
tgaacagacc cagctggaga 1260 tcgccacacg actgccaggc tcctattgtg
tgcaagtggc tgcagtcact ggagctggtg 1320 ctggagaact cagtacccct
gtctgcctcc ttttagagca ggccatggag caatcagcac 1380 gagaccccag
gaaacatgtt ccctggaccc tggaacagct gagggccacc ttgagacgac 1440
cagaagtcat tgccagtagt gctgtcctac tctggttgct gctactaggc attactgtgt
1500 gtatctacag acgacgcaaa gctggggtgc acctgggccc aggtctgtac
agatacacca 1560 gcgaggacgc cattctaaaa cacaggatgg accacagtga
ctccccatgg ctggcagaca 1620 cctggcgttc cacctctggc tctcgagacc
tgagcagcag cagcagcctt agtagtcggc 1680 tgggattgga ccctcgggac
ccactagagg gcaggcgctc cttgatctcc tgggaccctc 1740 ggagccccgg
tgtacccctg cttccagaca cgagcacgtt ttacggctcc ctcattgcag 1800
agcagccttc cagccctcca gtccggccaa gccccaagac accagctgct aggcgctttc
1860 catccaagtt ggctggaacc tccagcccct gggctagctc agatagtctc
tgcagccgca 1920 ggggactctg ttccccacgc atgtctctga cccctacaga
ggcttggaag gccaaaaaga 1980 agcaggaatt gcaccaagct aacagctccc
cactgctccg gggcagccac cccatggaaa 2040 tctgggcctg ggagttggga
agcagagcct ccaagaacct ttctcaaagc ccaggagaag 2100 cgccccgagc
cgtggtatcc tggcgtgctg tgggaccaca acttcaccgc aactccagtg 2160
agctggcatc tcgtccactc
cctccaacac ccctttctct tcgtggagct tccagtcatg 2220 acccacagag
ccagtgtgtg gagaagctcc aagctccctc ctctgaccca ctgccagcag 2280
cccctctctc cgtcctcaac tcttccagac cttccagccc ccaggcctct ttcctctcct
2340 gtcctagccc atcctccagc aacctgtcca gctcctcgct gtcatcctta
gaggaggagg 2400 aggatcagga cagcgtgctc acccccgagg aggtagccct
gtgtctggag ctcagtgatg 2460 gggaggagac acccacgaac agtgtatctc
ctatgccaag agctccttcc ccgccaacaa 2520 cctatggcta tatcagcata
ccaacctgct caggactggc agacatgggc agagctggcg 2580 ggggcgtggg
gtctgaggtt gggaacttac tgtatccacc tcggccctgc cccaccccta 2640
cacccagcga gggctccctg gccaatggtt ggggctcagc ttctgaggac aatgtcccca
2700 gcgccagggc cagcctggtt agctcttctg atggctcctt cctcgctgat
actcactttg 2760 ctcgtgccct ggcagtggct gtggatagct ttggcctcag
tctggatccc agggaagctg 2820 actgtgtctt cactgatgcc tcatcacctc
cctcccctcg gggtgatctc tccctgaccc 2880 gaagcttctc tctgcctttg
tgggagtgga ggccagactg gttggaagat gctgagatca 2940 gccacaccca
gaggctgggg agggggctgc ctccctggcc tcctgattct agggcctctt 3000
cccagcgaag ttggctaact ggtgctgtgc ccaaggctgg tgattcctcc tgaattgtcc
3060 ctgagaaggc cagaagagca cccagaccac tctcctgtct gtcccctggc
tttctcacat 3120 gtggaggtct tggcctatgc ttctctgtaa tagaagtcca
ccgtcactag gcttctggag 3180 agctctgtca ttgggattgt taaaataaat
gaaagcaaac caaaatatga tcacgggagt 3240 cttggattcc cactgagaac
aagacagcat cttcaggaca gcagactctc cacaaccaga 3300 acctttggcc
taagtaagcc tggctccgga gctcccacct aagtggatca tggaaagaag 3360
ggaagccaac caggtcttca ggaaggacag aaatgttttt tggtgagggc tatggtggag
3420 gacctgtgga agagccctct catatctact tggactcctc ccttagaggc
cagctcaacc 3480 ctttccccag tcacaccatg caaggaaact aaaggagaaa
ggtcgtggat gcagtgggcc 3540 ctatacagcg tcacagtcaa tgcttcaaag
tgagatcaat ggaggagact gaaggaaagg 3600 acgcagg 3607
* * * * *
References